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Genes coding for intermediate filament proteins closely related to the hagfish "thread keratins (TK)" alpha and gamma also exist in lamprey, teleosts and amphibians.

The "thread keratins (TK)" alpha and gamma so far have been considered highly specialized intermediate filament (IF) proteins restricted to hagfish. From lamprey, we now have sequenced five novel IF proteins closely related to TKalpha and TKgamma, respectively. Moreover, we have detected corresponding sequences in EST and genomic databases of teleosts and amphibians. The structure of the TKalpha genes and the positions of their deduced amino acid sequences in a phylogenetic tree clearly support their classification as type II keratins. The genes encoding TKgamma show a structure typical for type III IF proteins, whereas their positions in phylogenetic trees favor a close relationship to the type I keratins. Considering that most keratin-like sequences detected in the lancelet also exhibit a gene structure typical for type III IF proteins, it seems likely that the keratin gene(s) originated from an ancient type III IF protein gene. According to EST analyses, the expression of the thread keratins in teleost fish and amphibians may be particularly restricted to larval stages, which, in conjunction with the observed absence of TKalpha and TKgamma genes in any of the available Amniota databases, indicates a thread keratin function closely related to larval development in an aquatic environment.

Amino Acid Sequence↗

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.

Animals↗

A Twist in fate: evolutionary comparison of Twist structure and function.

The general requirement to induce mesoderm and allocate cells into different mesodermal tissues such as body muscle or heart is common in many animal embryos. Since the discovery of the twist gene, there has been great progress toward unraveling the molecular mechanisms that control mesoderm specification and differentiation. Twist was first identified in Drosophila as a gene crucial for proper gastrulation and mesoderm formation. In the fly embryo, Twist continues to play additional roles, allocating mesodermal cells into the body wall muscle fate and patterning a subset of these muscles. Twist is also required for proper differentiation of the adult musculature. Twist homologues have been identified in a great variety of organisms, which span the phylogenetic tree. These organisms include other invertebrates such as jellyfish, nematode, leech and lancelet as well as vertebrates such as frog, chick, fish, mouse and human. The Twist family shares both homology in structure across the basic helix-loop-helix domain and in expression during mesoderm and muscle development in most species. Here we review the current state of knowledge of the Twist family and consider how Twist functions during development. Moreover, we highlight experimental evidence that shows common themes that Twist employs during specification and patterning of the mesoderm among evolutionarily distant organisms. Conserved principles and the molecular mechanisms underlying them are discussed.

Amino Acid Sequence↗

Analysis of eight cDNAs and six genes for intermediate filament (IF) proteins in the cephalochordate Branchiostoma reveals differences in the IF multigene families of lower chordates and the vertebrates.

We report the sequences of seven new cytoplasmic intermediate filament (IF) proteins of the cephalochordate Branchiostoma. The eight sequences currently known describe four subfamilies (A, B, C and D). All eight IF proteins show the short-length version of the coil 1b subdomain found in vertebrates and lack the additional 42 residues present in all nuclear lamins and the protostomic IF proteins. Although the lancelet is considered to be the closest relative of the vertebrates, it is difficult to relate its IF subfamilies unambiguously to a particular type I-IV subfamily of vertebrates. C1 and C2 have tail domains with two 64 residue repeats of coiled coil-forming ability, a structural feature unknown for IF proteins from vertebrates or protostomia. The epidermal protein D1 shows only a slightly better identity score with vertebrate type II keratins than with type III proteins, but the D1 gene organization is that of type III proteins. The same holds for A1, A2, B1, B2 and C2 genes, although the latter has an additional and uniquely positioned intron. Antibodies (Ab) raised against recombinant C2 and D1 proteins reveal these proteins in epidermis, some internal epithelia and parts of the spinal cord. The results on exonic sequences, gene organization and expression suggest that Branchiostoma IF proteins may retain a largely archetypal condition, whereas the vertebrates have established the well-known type I-IV IF system.

Amino Acid Sequence↗

Tunicates and not cephalochordates are the closest living relatives of vertebrates.

Tunicates or urochordates (appendicularians, salps and sea squirts), cephalochordates (lancelets) and vertebrates (including lamprey and hagfish) constitute the three extant groups of chordate animals. Traditionally, cephalochordates are considered as the closest living relatives of vertebrates, with tunicates representing the earliest chordate lineage. This view is mainly justified by overall morphological similarities and an apparently increased complexity in cephalochordates and vertebrates relative to tunicates. Despite their critical importance for understanding the origins of vertebrates, phylogenetic studies of chordate relationships have provided equivocal results. Taking advantage of the genome sequencing of the appendicularian Oikopleura dioica, we assembled a phylogenomic data set of 146 nuclear genes (33,800 unambiguously aligned amino acids) from 14 deuterostomes and 24 other slowly evolving species as an outgroup. Here we show that phylogenetic analyses of this data set provide compelling evidence that tunicates, and not cephalochordates, represent the closest living relatives of vertebrates. Chordate monophyly remains uncertain because cephalochordates, albeit with a non-significant statistical support, surprisingly grouped with echinoderms, a hypothesis that needs to be tested with additional data. This new phylogenetic scheme prompts a reappraisal of both morphological and palaeontological data and has important implications for the interpretation of developmental and genomic studies in which tunicates and cephalochordates are used as model animals.

Animals↗

Amphioxus mitochondrial DNA, chordate phylogeny, and the limits of inference based on comparisons of sequences.

Analyses of both the nucleotide and amino acid sequences derived from all 13 mitochondrial protein-encoding genes (12,234 bp) of 19 metazoan species, including that of the lancelet Branchiostoma floridae ("amphioxus"), fail to yield the widely accepted phylogeny for chordates and, within chordates, for vertebrates. Given the breadth and the compelling nature of the data supporting that phylogeny, relationships supported by the mitochondrial sequence comparisons are almost certainly incorrect, despite their being supported by equally weighted parsimony, distance, and maximum-likelihood analyses. The incorrect groupings probably result in part from convergent base-compositional similarities among some of the taxa, similarities that are strong enough to overwhelm the historical signal. Comparisons among very distantly related taxa are likely to be particularly susceptible to such artifacts, because the historical signal is already greatly attenuated. Empirical results underscore the need for approaches to phylogenetic inference that go beyond simple site-by-site comparison of aligned sequences. This study and others indicate that, once a sequence sample of reasonable size has been obtained, accurate phylogenetic estimation may be better served by incorporating knowledge of molecular structures and processes into inference models and by seeking additional higher order characters embedded in those sequences, than by gathering ever larger sequence samples from the same organisms in he hope that the historical signal will eventually prevail.

Animals↗

Evolution and development of the chordates: collagen and pharyngeal cartilage.

Chordates evolved a unique body plan within deuterostomes and are considered to share five morphological characters, a muscular postanal tail, a notochord, a dorsal neural tube, an endostyle, and pharyngeal gill slits. The phylum Chordata typically includes three subphyla, Cephalochordata, Vertebrata, and Tunicata, the last showing a chordate body plan only as a larva. Hemichordates, in contrast, have pharyngeal gill slits, an endostyle, and a postanal tail but appear to lack a notochord and dorsal neural tube. Because hemichordates are the sister group of echinoderms, the morphological features shared with the chordates must have been present in the deuterostome ancestor. No extant echinoderms share any of the chordate features, so presumably they have lost these structures evolutionarily. We review the development of chordate characters in hemichordates and present new data characterizing the pharyngeal gill slits and their cartilaginous gill bars. We show that hemichordate gill bars contain collagen and proteoglycans but are acellular. Hemichordates and cephalochordates, or lancelets, show strong similarities in their gill bars, suggesting that an acellular cartilage may have preceded cellular cartilage in deuterostomes. Our evidence suggests that the deuterostome ancestor was a benthic worm with gill slits and acellular gill cartilages.

Animals↗

Gene duplication events producing muscle (M) and brain (B) isoforms of cytoplasmic creatine kinase: cDNA and deduced amino acid sequences from two lower chordates.

Creatine kinase (CK) is coded for by at least four loci in higher vertebrates--two cytoplasmic isoforms, muscle (M) and brain (B), and two mitochondrial isoforms, sarcomeric and ubiquitous. M is expressed primarily in skeletal muscle, while B is expressed in a variety of cells, including cardiac and smooth muscle fibers, neurons, transport epithelia, and photoreceptors. M and B subunits form very stable homodimers (MM [M-CK], BB [B-CK]) and heterodimers (MB). M-CK is capable of binding to the M line of the myofibril, thereby creating an energy transfer microcompartment; BB and MB CKs are not. M- and B-like CKs are present in all vertebrates yet examined, including fish. Cytoplasmic, dimeric CKs are widely distributed in the invertebrates. The only available amino acid sequence for an invertebrate dimeric CK, that of the protostome polychaete Chaetopterus variopedatus, is just as similar to the vertebrate M isoform as to the B isoform. Echinoderms lack dimeric, cytoplasmic CKs, which appear to be replaced by a dimeric arginine kinase which evolved secondarily from CK. Thus, it is likely that the gene duplication event producing the M and B isoforms occurred after the divergence of the chordates from echinoderms. To narrow down the timing of this duplication event, we obtained the cDNA and deduced amino acid sequences of dimeric CKs from the tunicate Ciona intestinalis (subphylum Urochordata) and the lancelet Branchiostoma floridae (subphylum Cephalochordata). Our results show that these CKs are strikingly similar to both invertebrate and vertebrate CKs. However, phylogenetic analyses by neighbor-joining and parsimony show that these two enzymes appeared to have diverged before the point of divergence of the M and B isoforms. Thus, the gene duplication event for formation of the muscle and brain isoforms of CK most likely occurred during the radiation of the fish, a time noted for gene duplication events at a variety of other loci.

Amino Acid Sequence↗

Evaluating hypotheses of deuterostome phylogeny and chordate evolution with new LSU and SSU ribosomal DNA data.

We investigated evolutionary relationships among deuterostome subgroups by obtaining nearly complete large-subunit ribosomal RNA (LSU rRNA)-gene sequences for 14 deuterostomes and 3 protostomes and complete small-subunit (SSU) rRNA-gene sequences for five of these animals. With the addition of previously published sequences, we compared 28 taxa using three different data sets (LSU only, SSU only, and combined LSU + SSU) under minimum evolution (with LogDet distances), maximum likelihood, and maximum parsimony optimality criteria. Additionally, we analyzed the combined LSU + SSU sequences with spectral analysis of LogDet distances, a technique that measures the amount of support and conflict within the data for every possible grouping of taxa. Overall, we found that (1) the LSU genes produced a tree very similar to the SSU gene tree, (2) adding LSU to SSU sequences strengthened the bootstrap support for many groups above the SSU-only values (e.g., hemichordates plus echinoderms as Ambulacraria; lancelets as the sister group to vertebrates), (3) LSU sequences did not support SSU-based hypotheses of pterobranchs evolving from enteropneusts and thaliaceans evolving from ascidians, and (4) the combined LSU + SSU data are ambiguous about the monophyly of chordates. No tree-building algorithm united urochordates conclusively with other chordates, although spectral analysis did so, providing our only evidence for chordate monophyly. With spectral analysis, we also evaluated several major hypotheses of deuterostome phylogeny that were constructed from morphological, embryological, and paleontological evidence. Our rRNA-gene analysis refutes most of these hypotheses and thus advocates a rethinking of chordate and vertebrate origins.

Animals↗

28S and 18S rDNA sequences support the monophyly of lampreys and hagfishes.

Resolving the interrelationships of three major extant lineages of vertebrates (hagfishes, lampreys, and gnathostomes) is a particularly important issue in evolution, because the basal resolution critically influences our understanding of primitive vertebrate characters. A consensus has emerged over the last 20 years that lampreys are the sister group to the gnathostomes and the hagfishes represent an ancient, basal lineage. This hypothesis has essentially displaced the classical hypothesis of monophyly of the cyclostomes (lampreys plus hagfishes). To test these hypotheses, we compared nearly complete ribosomal DNA sequences from each of these major lineages, as well as those from a cephalochordate and a urochordate, which represent a paraphyletic outgroup for assessing the basal vertebrate relationships. For this comparison, 92%-99% complete 28S rDNA sequences were obtained from the lancelet Branchiostoma floridae, the hagfish Eptatretus stouti, the lamprey Petromyzon marinus, and cartilaginous fishes Hydrolagus colliei and Squalus acanthias and were then analyzed with previously reported 28S and 18S rDNA sequences from other chordates. We conducted conventional (nonparametric) bootstrap analyses, under maximum-likelihood, parsimony, and minimum-evolution (using LogDet distances) criteria, of both 28S and 18S rDNA sequences considered separately and combined. All these analyses provide moderate to very strong support for the monophyly of the cyclostomes. Furthermore, the currently accepted hypothesis of a lamprey-gnathostome clade is moderately rejected by the Kishino-Hasegawa test (P = 0.099) and resoundingly rejected by parametric bootstrap tests (P < 0.01) in favor of monophyly of living cyclostomes. Another significant finding is that the hagfish E. stouti has the longest 28S rDNA gene known in any organism (> 5,200 nt).

Animals↗

Complete sequence, gene arrangement, and genetic code of mitochondrial DNA of the cephalochordate Branchiostoma floridae (Amphioxus)

We have determined the 15,083-nucleotide (nt) sequence of the mitochondrial DNA (mtDNA) of the lancelet Branchiostoma floridae (Chordata: Cephalochordata). As is typical in metazoans, the mtDNA encodes 13 protein, 2 rRNA, and 22 tRNA genes. The gene arrangement differs from the common vertebrate arrangement by only four tRNA gene positions. Three of these are unique to Branchiostoma, but the fourth is in a position that is primitive for chordates. It shares the genetic code variations found in vertebrate mtDNAs except that AGA = serine, a code variation found in many invertebrate phyla but not in vertebrates (the related codon AGG was not found). Branchiostoma mtDNA lacks a vertebrate-like control region; its largest noncoding region (129 nt) is unremarkable in sequence or base composition, and its location between ND5 and tRNAG differs from that usually found in vertebrates. It also lacks a potential hairpin DNA structure like those found in many (though not in all) vertebrates to serve as the second-strand (i.e., L-strand) origin of replication. Perhaps related to this, the sequence corresponding to the DHU arm of tRNAC cannot form a helical stem, a condition found in a few other vertebrate mtDNAs that also lack a canonical L-strand origin of replication. ATG and GTG codons appear to initiate translation in 11 and 2 of the protein-encoding genes, respectively. Protein genes end with complete (TAA or TAG) or incomplete (T or TA) stop codons; the latter are presumably converted to TAA by post-transcriptional polyadenylation.

Animals↗

Evolutionary transformations of myoseptal tendons in gnathostomes.

Axial undulations in fishes are powered by a series of three-dimensionally folded myomeres separated by sheets of connective tissue, the myosepta. Myosepta have been hypothesized to function as transmitters of muscular forces to axial structures during swimming, but the difficulty of studying these delicate complex structures has precluded a more complete understanding of myoseptal mechanics. We have developed a new combination of techniques for visualizing the three-dimensional morphology of myosepta, and here we present their collagen-fibre architecture based on examination of 62 species representing all of the major clades of notochordates. In all gnathostome fishes, each myoseptum bears a set of six specifically arranged tendons. Because these tendons are not present outside the gnathostomes (i.e. they are absent from lampreys, hagfishes and lancelets), they represent evolutionary novelties of the gnathostome ancestor. This arrangement has remained unchanged throughout 400 Myr of gnathostome evolution, changing only on the transition to land. The high uniformity of myoseptal architecture in gnathostome fishes indicates functional significance and may be a key to understanding general principles of fish swimming mechanics. In the design of future experiments or biomechanical models, myosepta have to be regarded as tendons that can distribute forces in specific directions.

Animals↗

Invertebrate immune systems--not homogeneous, not simple, not well understood.

The approximate 30 extant invertebrate phyla have diversified along separate evolutionary trajectories for hundreds of millions of years. Although recent work understandably has emphasized the commonalities of innate defenses, there is also ample evidence, as from completed genome studies, to suggest that even members of the same invertebrate order have taken significantly different approaches to internal defense. These data suggest that novel immune capabilities will be found among the different phyla. Many invertebrates have intimate associations with symbionts that may play more of a role in internal defense than generally appreciated. Some invertebrates that are either long lived or have colonial body plans may diversify components of their defense systems via somatic mutation. Somatic diversification following pathogen exposure, as seen in plants, has been investigated little in invertebrates. Recent molecular studies of sponges, cnidarians, shrimp, mollusks, sea urchins, tunicates, and lancelets have found surprisingly diversified immune molecules, and a model is presented that supports the adaptive value of diversified non-self recognition molecules in invertebrates. Interactions between invertebrates and viruses also remain poorly understood. As we are in the midst of alarming losses of coral reefs, increased pathogen challenge to invertebrate aquaculture, and rampant invertebrate-transmitted parasites of humans and domestic animals, we need a better understanding of invertebrate immunology.

Animals↗

Evidence from 18S ribosomal RNA sequences that lampreys and hagfishes form a natural group.

Lampreys and hagfishes (cyclostomes) traditionally were considered to be a natural (monophyletic) group. Recently, the consensus of opinion, based largely on morphological analyses, has shifted to a view that lampreys are more closely related to jawed vertebrates (gnathostomes) than to hagfishes. Phylogenetic comparisons of 18S ribosomal RNA sequences from two hagfishes, two lampreys, a tunicate, a lancelet, and a number of gnathostomes support the monophyly of the cyclostomes. These data force a reassessment of several features of early vertebrate evolution.

Animals↗

"Functional polarity" of the spinal cord caused by its longitudinal electric dipole moment.

Specific reactions of the vertebrates that occur through lengthwise application of direct-current (DC) electric fields in the spinal cord and are dependent on the direction of the current (e.g., electric narcosis and electric excitation) can be explained by the existence of a longitudinal dipole moment in the spinal cord. The relationship between "functional polarity of the spinal cord" and its longitudinal dipole moment was established in two different experimental series. 1) Reactions of functional polarity were quantitatively tested on Amphioxus (lancelet) and on numerous Teleostei. On this point the findings of other researchers were confirmed and complemented. 2) Fresh, almost-live spinal cord preparations (cow) were quantitatively tested using pyroelectric (PE) methods. A longitudinal electric dipole moment was demonstrated along the entire length of the white matter. Moreover fresh spinal cord preparations showed a polar orientation on sudden temperature change in a high-voltage DC field. The PE behavior showed a pronounced electric field dependence. Because the white matter is largely composed of longitudinally arranged nerve fibers, individual axons may also have a longitudinal electric dipole moment. Consequently one might expect the following: polar orientation of the growing neurite by an applied DC field, an electric dipole field inside and outside the axon, and a transport of charge carriers along the lines of flux of this dipole field. Moreover PE and piezoelectric (PZE) properties of the neuron should be provable.

Animals↗

Basal jawed vertebrate phylogeny inferred from multiple nuclear DNA-coded genes.

BACKGROUND: Phylogenetic analyses of jawed vertebrates based on mitochondrial sequences often result in confusing inferences which are obviously inconsistent with generally accepted trees. In particular, in a hypothesis by Rasmussen and Arnason based on mitochondrial trees, cartilaginous fishes have a terminal position in a paraphyletic cluster of bony fishes. No previous analysis based on nuclear DNA-coded genes could significantly reject the mitochondrial trees of jawed vertebrates. RESULTS: We have cloned and sequenced seven nuclear DNA-coded genes from 13 vertebrate species. These sequences, together with sequences available from databases including 13 jawed vertebrates from eight major groups (cartilaginous fishes, bichir, chondrosteans, gar, bowfin, teleost fishes, lungfishes and tetrapods) and an outgroup (a cyclostome and a lancelet), have been subjected to phylogenetic analyses based on the maximum likelihood method. CONCLUSION: Cartilaginous fishes have been inferred to be basal to other jawed vertebrates, which is consistent with the generally accepted view. The minimum log-likelihood difference between the maximum likelihood tree and trees not supporting the basal position of cartilaginous fishes is 18.3 +/- 13.1. The hypothesis by Rasmussen and Arnason has been significantly rejected with the minimum log-likelihood difference of 123 +/- 23.3. Our tree has also shown that living holosteans, comprising bowfin and gar, form a monophyletic group which is the sister group to teleost fishes. This is consistent with a formerly prevalent view of vertebrate classification, although inconsistent with both of the current morphology-based and mitochondrial sequence-based trees. Furthermore, the bichir has been shown to be the basal ray-finned fish. Tetrapods and lungfish have formed a monophyletic cluster in the tree inferred from the concatenated alignment, being consistent with the currently prevalent view. It also remains possible that tetrapods are more closely related to ray-finned fishes than to lungfishes.

Animals↗

Morphometric partitioning of respiratory surfaces in amphioxus (Branchiostoma lanceolatum Pallas).

The anatomical diffusing factors (ADFs), defined as the ratio of surface area to the thickness of the diffusion barrier, of possible respiratory surfaces of adult amphioxus (Branchiostoma lanceolatum) were evaluated using stereological methods. The ADF is greatest for the lining of the atrium and for the skin covering the segmental muscles. Calculation of the diffusing capacities for O(2) revealed that the lining of the atrium makes up nearly 83 % of the entire diffusing capacity (8.86 x 10(-3) microl min(-1)mg(-1)kPa(-1) while the skin over the segmental muscles (9%), the skin over the metapleural fold (4%) and the gill bars (4%) are of minor importance. The diffusing capacity of surfaces lying over coelomic cavities makes up 76% of the whole diffusing capacity, which is consistent with the hypothesis that the coelom may function as a circulatory system for respiratory gases. Muscles have approximately 23% of the entire diffusing capacity, indicating that they may be self-sufficient for O(2) uptake. The diffusing capacity of the blood vessels in the gill bars is only 1% of the total. Thus, the 'gills' lack significant function as respiratory organs in amphioxus (lancelets).

Animals↗

Novel acetylcholinesterase target site for malaria mosquito control.

Current anticholinesterase pesticides were developed during World War II and are toxic to mammals because they target a catalytic serine residue of acetylcholinesterases (AChEs) in insects and in mammals. A sequence analysis of AChEs from 73 species and a three-dimensional model of a malaria-carrying mosquito (Anopheles gambiae) AChE (AgAChE) reported here show that C286 and R339 of AgAChE are conserved at the opening of the active site of AChEs in 17 invertebrate and four insect species, respectively. Both residues are absent in the active site of AChEs of human, monkey, dog, cat, cattle, rabbit, rat, and mouse. The 17 invertebrates include house mosquito, Japanese encephalitis mosquito, African malaria mosquito, German cockroach, Florida lancelet, rice leaf beetle, African bollworm, beet armyworm, codling moth, diamondback moth, domestic silkworm, honey bee, oat or wheat aphid, the greenbug, melon or cotton aphid, green peach aphid, and English grain aphid. The four insects are house mosquito, Japanese encephalitis mosquito, African malaria mosquito, and German cockroach. The discovery of the two invertebrate-specific residues enables the development of effective and safer pesticides that target the residues present only in mosquito AChEs rather than the ubiquitous serine residue, thus potentially offering an effective control of mosquito-borne malaria. Anti-AgAChE pesticides can be designed to interact with R339 and subsequently covalently bond to C286. Such pesticides would be toxic to mosquitoes but not to mammals.

Acetylcholinesterase↗