Methylation of genomes and genes at the invertebrate-vertebrate boundary.

Patterns of DNA methylation in animal genomes are known to vary from an apparent absence of modified bases, via methylation of a minor fraction of the genome, to genome-wide methylation. Representative genomes from 10 invertebrate phyla comprise predominantly nonmethylated DNA and (usually but not always) a minor fraction of methylated DNA. In contrast, all 27 vertebrate genomes that have been examined display genome-wide methylation. Our studies of chordate genomes suggest that the transition from fractional to global methylation occurred close to the origin of vertebrates, as amphioxus has a typically invertebrate methylation pattern whereas primitive vertebrates (hagfish and lamprey) have patterns that are typical of vertebrates. Surprisingly, methylation of genes preceded this transition, as many invertebrate genes have turned out to be heavily methylated. Methylation does not preferentially affect genes whose expression is highly regulated, as several housekeeping genes are found in the heavily methylated fraction whereas several genes expressed in a tissue-specific manner are in the nonmethylated fraction.     

Evolution of the reproductive endocrine system in chordates

The cephalochordate, amphioxus, is phylogenetically placed at the most primitive position in the chordate clade. Despite many studies on the endocrine system of amphioxus, definitive evidence has not been reported for the presence an endocrine system comparable to the pituitary-gonadal axis, which is important in the regulation of reproduction in vertebrates. Recent genome analyses in the amphioxus, Branchiostoma floridae, showed that it does not have any pituitary hormone genes except the thyrostimulin gene. Thyrostimulin is a heterodimeric glycoprotein hormone consisting of α and β subunits, and is present in various organs of vertebrates. Analyses of a phylogenetic tree and a synteny suggest that amphioxus' thyrostimulin is an ancestral type of the glycoprotein hormones in chordates. In addition, genes for sex steroidogenic enzymes belonging to the CYP family were found in the genome sequences. The conversion pathway of sex steroids from cholesterol to estrogen, androgen, and major sex steroids was also identified in the gonads of amphioxus in vitro. Furthermore, we demonstrated the expression of genes encoding thyrostimulin and sex steroidogenic enzymes by an in situ hybridization technique. Here, we discuss the evolution of hormones and reproductive functions in the neuroendocrine control system of chordates.     

"Insights of early chordate genomics: endocrinology and development in amphioxus, tunicates and lampreys": introduction to the symposium

This symposium focused on the evolution of chordate genomes, in particular, those events that occurred before the appearance of jawed vertebrates. The aim was to highlight insights that have come from the genome sequences of jawless chordates (lampreys, tunicates, and amphioxus) not only into evolution of chordate genomes, but also into the evolution of the organism. To this end, we brought together researchers whose recent work on these organisms spans the gap from genomics to the evolution of body forms and functions as exemplified by endocrine systems and embryonic development.     

Amphioxus: a peaceful anchovy fillet to illuminate Chordate Evolution (II)

The genome of the amphioxus is on the horizon. With Linda Holland and Jeremy Gibson-Brown at the forefront, with all the amphioxus community behind, and with the Joint Genome Institute, the amphioxus genome will see the light this year, 2006. Hope that it will reflect the "prototypical" preduplicative genome of vertebrates. It may answer definitively what the human genome did not: Are we vertebrates octaploid? Will it shed light on the novelties that helped non-chordates to be chordates? And more, will amphioxus, with a simpler genome, be developed to a senior "experimental model system", allowing the testing of molecular functions in a non-duplicated genome background and allowing genetic modification to "recapitulate" evolution? Thanks to an outstanding collaboration between labs, the laboratory culture of amphioxus is underway after years of hard work in the field. 2007 looks promising for amphioxus research.     

Presence and role of cytosine methylation in DNA viruses of animals

Nucleotide composition varies greatly among DNA viruses of animals, yet the evolutionary pressures and biological mechanisms driving these patterns are unclear. One of the most striking discrepancies lies in the frequency of CpG (the dinucleotide CG, linked by a phosphate group), which is underrepresented in most small DNA viruses (those with genomes below 10 kb) but not in larger DNA viruses. Cytosine methylation might be partially responsible, but research on this topic has focused on a few virus groups. For several viruses that integrate their genome into the host genome, the methylation status during this stage has been studied extensively, and the relationship between methylation and viral-induced tumor formation has been examined carefully. However, for actively replicating viruses—particularly small DNA viruses—the methylation status of CpG motifs is rarely known and the effects on the viral life cycle are obscure. In vertebrate host genomes, most cytosines at CpG sites are methylated, which in vertebrates acts to regulate gene expression and facilitates the recognition of unmethylated, potentially pathogen-associated DNA. Here we briefly introduce cytosine methylation before reviewing what is currently known about CpG methylation in DNA viruses.     

Enhancer evolution in chordates: Lessons from functional analyses of cephalochordate cis-regulatory modules

Chordates comprise three major groups, cephalochordates (amphioxus), tunicates (urochordates), and vertebrates. Since cephalochordates were the early branching group, comparisons between amphioxus and other chordates help us to speculate about ancestral chordates. Here, I summarize accumulating data from functional studies analyzing amphioxus cis-regulatory modules (CRMs) in model systems of other chordate groups, such as mice, chickens, clawed frogs, fish, and ascidians. Conservatism and variability of CRM functions illustrate how gene regulatory networks have evolved in chordates. Amphioxus CRMs, which correspond to CRMs deeply conserved among animal phyla, govern reporter gene expression in conserved expression domains of the putative target gene in host animals. In addition, some CRMs located in similar genomic regions (intron, upstream, or downstream) also possess conserved activity, even though their sequences are divergent. These conservative CRM functions imply ancestral genomic structures and gene regulatory networks in chordates. However, interestingly, if expression patterns of amphioxus genes do not correspond to those of orthologs of experimental models, some amphioxus CRMs recapitulate expression patterns of amphioxus genes, but not those of endogenous genes, suggesting that these amphioxus CRMs are close to the ancestral states of chordate CRMs, while vertebrates/tunicates innovated new CRMs to reconstruct gene regulatory networks subsequent to the divergence of the cephalochordates. Alternatively, amphioxus CRMs may have secondarily lost ancestral CRM activity and evolved independently. These data help to solve fundamental questions of chordate evolution, such as neural crest cells, placodes, a forebrain/midbrain, and genome duplication. Experimental validation is crucial to verify CRM functions and evolution.     

Actors of the tyrosine kinase receptor downstream signaling pathways in amphioxus

SUMMARY One of the major goals of evo-developmentalists is to understand how the genetic mechanisms controlling embryonic development have evolved to create the current diversity of bodyplans that we encounter in the animal kingdom. Tyrosine kinase receptors (RTKs) are transmembrane receptors present in all metazoans known to control several developmental processes. They act via the activation of various cytoplasmic signaling cascades, including the mitogen-activated protein kinase (MAPK), the PI3K/Akt, and the phospholipase C-γ (PLCγ)/protein kinase C (PKC) pathways. In order to address the evolution of these three pathways and their involvement during embryogenesis in chordates, we took advantage of the complete genome sequencing of a key evolutionarily positioned species, the cephalochordate amphioxus, and searched for the complete gene set of the three signaling pathways. We found that the amphioxus genome contains all of the most important modules of the RTK-activated cascades, and looked at the embryonic expression of two genes selected from each cascade. Our data suggest that although the PI3K/Akt pathway may have ubiquitous functions, the MAPK and the PLCγ/PKC cascades may play specific roles in amphioxus development. Together with data known in vertebrates, the expression pattern of PKC in amphioxus suggests that the PLCγ/PKC cascade was implicated in neural development in the ancestor of all chordates.     

The amphioxus Hairy family: differential fate after duplication

Vertebrate Hairy genes are highly pleiotropic and have been implicated in numerous functions, such as somitogenesis, neurogenesis and endocrine tissue development. In order to gain insight into the timing of acquisition of these roles by the Hairy subfamily, we have cloned and studied the expression pattern of the Hairy gene(s) in amphioxus. The cephalochordate amphioxus is widely believed to be the living invertebrate more closely related to vertebrates, the genome of which has not undergone the massive gene duplications that took place early during vertebrate evolution. Surprisingly, we have isolated eight Hairy genes from the 'pre-duplicative' amphioxus genome. In situ hybridisation on amphioxus embryos showed that Hairy genes had experienced a process of subfunctionalisation that is predicted in the DDC model (for duplication-degeneration-complementation). Only the summation of four out of the eight Amphi-Hairy genes expression resembles the expression pattern of vertebrate Hairy genes, i.e. in the central nervous system, presomitic mesoderm, somites, notochord and gut. In addition, Amphi-Hairy genes expression suggest that amphioxus early somites are molecularly prefigured in an anteroposterior sequence in the dorsolateral wall of the archenteron, and the presence of a midbrain/hindbrain boundary. The expansion of the amphioxus Hairy subfamily request for caution when deducing the evolutionary history of a gene family in chordates based in the singularity of the amphioxus genome. Amphioxus may resemble the ancestor of the vertebrates, but it is not the ancestor, only its closest living relative, a privileged position that should not assume the freezing of its genome.     

Evolution of the genetic machinery of the visual cycle: a novelty of the vertebrate eye?

The discovery in invertebrates of ciliary photoreceptor cells and ciliary (c)-opsins established that at least two of the three elements that characterize the vertebrate photoreceptor system were already present before vertebrate evolution. However, the origin of the third element, a series of biochemical reactions known as the "retinoid cycle," remained uncertain. To understand the evolution of the retinoid cycle, I have searched for the genetic machinery of the cycle in invertebrate genomes, with special emphasis on the cephalochordate amphioxus. Amphioxus is closely related to vertebrates, has a fairly prototypical genome, and possesses ciliary photoreceptor cells and c-opsins. Phylogenetic and structural analyses of the amphioxus sequences related with the vertebrate machinery do not support a function of amphioxus proteins in chromophore regeneration but suggest that the genetic machinery of the retinoid cycle arose in vertebrates due to duplications of ancestral nonvisual genes. These results favor the hypothesis that the retinoid cycle machinery was a functional innovation of the primitive vertebrate eye.     

Essential role of Bmp signaling and its positive feedback loop in the early cell fate evolution of chordates

In chordates, early separation of cell fate domains occurs prior to the final specification of ectoderm to neural and non-neural as well as mesoderm to dorsal and ventral during development. Maintaining such division with the establishment of an exact border between the domains is required for the formation of highly differentiated structures such as neural tube and notochord. We hypothesized that the key condition for efficient cell fate separation in a chordate embryo is the presence of a positive feedback loop for Bmp signaling within the gene regulatory network (GRN), underlying early axial patterning. Here, we therefore investigated the role of Bmp signaling in axial cell fate determination in amphioxus, the basal chordate possessing a centralized nervous system. Pharmacological inhibition of Bmp signaling induces dorsalization of amphioxus embryos and expansion of neural plate markers, which is consistent with an ancestral role of Bmp signaling in chordate axial patterning and neural plate formation. Furthermore, we provided evidence for the presence of the positive feedback loop within the Bmp signaling network of amphioxus. Using mRNA microinjections we found that, in contrast to vertebrate Vent genes, which promote the expression of Bmp4, amphioxus Vent1 is likely not responsible for activation of cephalochordate ortholog Bmp2/4. Cis-regulatory analysis of amphioxus Bmp2/4, Admp and Chordin promoters in medaka embryos revealed remarkable conservation of the gene regulatory information between vertebrates and basal chordates. Our data suggest that emergence of a positive feedback loop within the Bmp signaling network may represent a key molecular event in the evolutionary history of the chordate cell fate determination.     

The amphioxus genome enlightens the evolution of the thyroid hormone signaling pathway

Thyroid hormones (THs) have pleiotropic effects on vertebrate development, with amphibian metamorphosis as the most spectacular example. However, developmental functions of THs in non-vertebrate chordates are largely hypothetical and even TH endogenous production has been poorly investigated. In order to get better insight into the evolution of the thyroid hormone signaling pathway in chordates, we have taken advantage of the recent release of the amphioxus genome. We found amphioxus homologous sequences to most of the genes encoding proteins involved in thyroid hormone signaling in vertebrates, except the fast-evolving thyroglobulin: sodium iodide symporter, thyroid peroxidase, deiodinases, thyroid hormone receptor, TBG, and CTHBP. As only some genes encoding proteins involved in TH synthesis regulation were retrieved (TRH, TSH receptor, and CRH receptor but not their corresponding receptors and ligands), there may be another mode of upstream regulation of TH synthesis in amphioxus. In accord with the notion that two whole genome duplications took place at the base of the vertebrate tree, one amphioxus gene often corresponded to several vertebrate homologs. However, some amphioxus specific duplications occurred, suggesting that several steps of the TH pathway were independently elaborated in the cephalochordate and vertebrate lineages. The present results therefore indicate that amphioxus is capable of producing THs. As several genes of the TH signaling pathway were also found in the sea urchin genome, we propose that the thyroid hormone signaling pathway is of ancestral origin in chordates, if not in deuterostomes, with specific elaborations in each lineage, including amphioxus.     

Evolution of the Role of RA and FGF Signals in the Control of Somitogenesis in Chordates

During vertebrate development, the paraxial mesoderm becomes segmented, forming somites that will give rise to dermis, axial skeleton and skeletal muscles. Although recently challenged, the "clock and wavefront" model for somitogenesis explains how interactions between several cell-cell communication pathways, including the FGF, RA, Wnt and Notch signals, control the formation of these bilateral symmetric blocks. In the cephalochordate amphioxus, which belongs to the chordate phylum together with tunicates and vertebrates, the dorsal paraxial mesendoderm also periodically forms somites, although this process is asymmetric and extends along the whole body. It has been previously shown that the formation of the most anterior somites in amphioxus is dependent upon FGF signalling. However, the signals controlling somitogenesis during posterior elongation in amphioxus are still unknown. Here we show that, contrary to vertebrates, RA and FGF signals act independently during posterior elongation and that they are not mandatory for posterior somites to form. Moreover, we show that RA is not able to buffer the left/right asymmetry machinery that is controlled through the asymmetric expression of Nodal pathway actors. Our results give new insights into the evolution of the somitogenesis process in chordates. They suggest that RA and FGF pathways have acquired specific functions in the control of somitogenesis in vertebrates. We propose that the "clock and wavefront" system was selected specifically in vertebrates in parallel to the development of more complex somite-derived structures but that it was not required for somitogenesis in the ancestor of chordates.     

Comparative Genomic Study Reveals a Transition from TA Richness in Invertebrates to GC Richness in Vertebrates at CpG Flanking Sites： An Indication for Context-Dependent Mutagenicity of Methylated CpG Sites

Vertebrate genomes are characterized with CpG deficiency, particularly for GCpoor regions. The GC content-related CpG deficiency is probably caused by context-dependent deamination of methylated CpG sites. This hypothesis was examined in this study by comparing nucleotide frequencies at CpG flanking positions among invertebrate and vertebrate genomes. The finding is a transition of nucleotide preference of 5＇ T to 5＇ A at the invertebrate-vertebrate boundary, indicating that a large number of CpG sites with 5＇ Ts were depleted because of global DNA methylation developed in vertebrates. At genome level, we investigated CpG observed/expected （obs/exp） values in 500 bp fragments, and found that higher CpG obs/exp value is shown in GC-poor regions of invertebrate genomes （except sea urchin） but in GC-rich sequences of vertebrate genomes. We next compared GC content at CpG flanking positions with genomic average, showing that the GC content is lower than the average in invertebrate genomes, but higher than that in vertebrate genomes. These results indicate that although 5＇ T and 5＇ A are different in inducing deamination of methylated CpG sites, GC content is even more important in affecting the deamination rate. In all the tests, the results of sea urchin are similar to vertebrates perhaps due to its fractional DNA methylation. CpG deficiency is therefore suggested to be mainly a result of high mutation rates of methylated CpG sites in GC-poor regions.     

DNA Sequence Explains Seemingly Disordered Methylation Levels in Partially Methylated Domains of Mammalian Genomes

For the most part metazoan genomes are highly methylated and harbor only small regions with low or absent methylation. In contrast, partially methylated domains (PMDs), recently discovered in a variety of cell lines and tissues, do not fit this paradigm as they show partial methylation for large portions (20%–40%) of the genome. While in PMDs methylation levels are reduced on average, we found that at single CpG resolution, they show extensive variability along the genome outside of CpG islands and DNase I hypersensitive sites (DHS). Methylation levels range from 0% to 100% in a roughly uniform fashion with only little similarity between neighboring CpGs. A comparison of various PMD-containing methylomes showed that these seemingly disordered states of methylation are strongly conserved across cell types for virtually every PMD. Comparative sequence analysis suggests that DNA sequence is a major determinant of these methylation states. This is further substantiated by a purely sequence based model which can predict 31% (R²) of the variation in methylation. The model revealed CpG density as the main driving feature promoting methylation, opposite to what has been shown for CpG islands, followed by various dinucleotides immediately flanking the CpG and a minor contribution from sequence preferences reflecting nucleosome positioning. Taken together we provide a reinterpretation for the nucleotide-specific methylation levels observed in PMDs, demonstrate their conservation across tissues and suggest that they are mainly determined by specific DNA sequence features.     

Epigenetics: differential DNA methylation in mammalian somatic tissues

Epigenetics refers to heritable phenotypic alterations in the absence of DNA sequence changes, and DNA methylation is one of the extensively studied epigenetic alterations. DNA methylation is an evolutionally conserved mechanism to regulate gene expression in mammals. Because DNA methylation is preserved during DNA replication it can be inherited. Thus, DNA methylation could be a major mechanism by which to produce semi-stable changes in gene expression in somatic tissues. Although it remains controversial whether germ-line DNA methylation in mammalian genomes is stably heritable, frequent tissue-specific and disease-specific de novo methylation events are observed during somatic cell development/differentiation. In this minireview, we discuss the use of restriction landmark genomic scanning, together with in silico analysis, to identify differentially methylated regions in the mammalian genome. We then present a rough overview of quantitative DNA methylation patterns at 4600 NotI sites and more than 150 differentially methylated regions in several C57BL/6J mouse tissues. Comparative analysis between mice and humans suggests that some, but not all, tissue-specific differentially methylated regions are conserved. A deeper understanding of cell-type-specific differences in DNA methylation might lead to a better illustration of the mechanisms behind tissue-specific differentiation in mammals.     

Evidence for the evolution of tenascin and fibronectin early in the chordate lineage

Fibronectin and tenascin are extracellular matrix glycoproteins that play important roles in cell adhesion and motility. In a previous study we provided evidence that tenascin first appeared early in the chordate lineage. As tenascin has been proposed to act, in part, through modulation of cell-fibronectin interactions, we sought here to identify fibronectin genes in non-vertebrate chordates and other invertebrates to determine if tenascin and fibronectin evolved separately or together, and to identify phylogenetically conserved features of both proteins. We found that the genome of the urochordate Ciona savignyi contains both a tenascin gene and a gene encoding a fibronectin-like protein with fibronectin type 1, 2 and 3 repeats. The genome of the cephalochordate Branchiostoma floridae (amphioxus) also has a tenascin gene. However, we could not identify a fibronectin-like gene in B. floridae, nor could we identify fibronectin or tenascin genes in echinoderms, protostomes or cnidarians. If urochordates are more closely related to vertebrates, tenascin may have evolved before fibronectin in an ancestor common to tunicates and amphioxus. Alternatively, tenascin and fibronectin may have evolved in an ancestor common to B. floridae and C. savignyi and the fibronectin gene was subsequently lost in the cephalochordate lineage. The fibronectin-like gene from C. savignyi does not encode the RGD motif for integrin binding found in all vertebrate fibronectins, and it lacks most of the fibronectin type 1 domains believed to be critical for fibrillogenesis. In contrast, the tenascin gene in B. floridae encodes multiple RGD motifs, suggesting that integrin binding is fundamental to tenascin function.     

Temporal and regional changes in DNA methylation in the embryonic, extraembryonic and germ cell lineages during mouse embryo development

This paper shows stage- and tissue-specific global demethylation and remethylation occurring during embryonic development. The egg genome is strikingly undermethylated and the sperm genome relatively methylated. Following a loss of genomic methylation during preimplantation development, embryonic and extraembryonic lineages are progressively and independently methylated to different final extents. Methylation continues postgastrulation and hence could be a mechanism initiating, or confirming, differential programming in the definitive germ layers. It is proposed that much of the methylation observed in somatic tissues acts to stabilize and reinforce prior events that regulate the activity of specific genes, chromosome domains or the X chromosome (in females). Fetal germ cell DNA is markedly undermethylated and we favour the idea that the germ lineage is set aside before the occurrence of extensive methylation of DNA in fetal precursor cells.