Review by Simon House, in the book: 'Handbook of Epigenetics'*, which includes significant description of the work of Professor Michael Crawford and David Marsh.
* Handbook of Epigenetics: The New Molecular and Medical Genetics [Hardcover], Editor: Trygve Tollefsbol, Publisher: Academic Press; 1 edition (21 Oct 2010), ISBN-10: 0123757096, ISBN-13: 978-0123757098.
Epigenetics in Adaptive Evolution and Development: The interplay between evolving species and epigenetic mechanisms
Simon H. House*
*Declaration – I declare I have no conflict of interest.
Pressure for molecular evidenceAs research advances rapidly the definition of “epigenetics” evolves, but a definition currently acknowledged widely is: “changes in phenotype (appearance) or gene expression caused by mechanisms other than changes in the underlying DNA sequence” (1).
Practical indications of epigenetics
Epigenetics is more than a fascinating and fast burgeoning field of biological research: it is of vital consequence to the human race as we have come into arguably the worst crises in new forms of disease the human race has encountered. These include the “non-communicable” diseases related to the metabolic syndrome. Obesity, diabetes, cardiovascular and mental health disorders are increasingly recognized as connected with epigenetic changes of early origin.
One of the first scientists to prophesy specific permanent effects on health of nutrition from before conception and during development was Professor Michael A. Crawford, Director of the Institute of Brain Chemistry and Human Nutrition. In 1972 he related his and others’ findings to the effects of current nutrition on our current evolution as well as health, with particular emphasis on preconception nutrition, and on marine omega-3 oils in brain and heart development (2,3,4). Such lifelong effects of fetal and infant nutrition are better known by the term “The Barker Hypothesis” (5,6). Crawford used the adjective “epigenetic” in a broad sense, but although the term “epigenetics” had already been coined by Conrad Waddington in 1937 to describe environmental effects on the phenotype, molecular comprehension was delayed for half a century.
Nutrition is one aspect of environment readily studied objectively. Less amenable to objective study are stressors and stress, particularly the lasting impacts of maternal–fetal stress. Nonetheless remarkable evidence has come from work of pre- and perinatal societies, and notably of Dr Frank Lake, between 1954 and 1982, (7,8,9) instanced in the section below, “Continuing evolutionary impacts”. This evidence is becoming more intelligible through epigenetics.
Environment and evolution
Crawford’s proclaiming of the powerful effects of diet on development, culminated in his insistence that only at the waterside could the human species have achieved so large a brain, sustained by plentiful fish and shellfish with their essential marine oils, docosahexaenoic and eicosapentaenoic acids (DHA and EPA). The only other mammals to retain a large brain as they became at least as large were marine, such as dolphins and whales. Not until the 1990s was the power of Crawford’s case acknowledged by leaders in paleontology, having re-dated remains with electron-spin technology, and related them to evidence of contemporary water levels (10,11). Michael Crawford and David Marsh (12,13) had emphasized that Darwin, in The Origin of the Species (14), had attributed adaptation to “Conditions of Existence”, as a higher law than “Unity of Type”. The former was compatible with Lamarck’s (15) first basic law that “organs, thus species, change in response to a need created by a changing environment.” His second law that “such change was passed through the hereditary mechanism to the offspring” was intrinsic to Darwin’s “Unity of Type”, even if Darwin made more of random change. Sifted from the theistic and teleological elements, these two scientists’ theories stand the light of current science (16).
The commonly held view that adaptation was by natural selection of merely random changes, the “neo-Darwinist” view, is attributable to “the Weismann barrier” (17) – there are even scientists who use the term “Darwinism” clearly meaning “neo-Darwinism”. Although in his introduction Darwin writes, “I am convinced that Natural Selection has been the main, but not exclusive, means of modification”; concluding Chapter 6 he affirms:
“The expression of conditions of existence is fully embraced by natural selection [which acts by] adapting the varying parts of each being to its organic and inorganic conditions of life”. He adds “[Of the] two great laws – Unity of Type, and the Conditions of Existence? the law of the Conditions of Existence is the higher law; as it includes, through the inheritance of former variations and adaptations, that of Unity of Type.”
Darwin goes on:
“I can see no very great difficulty? in believing that natural selection has converted the simple apparatus of an optic nerve merely coated with pigment and invested by transparent membrane, into an optical instrument as perfect as is possessed by any member of the great Articulate class (nor) that natural selection has actually converted a swimbladder into a lung, or organ used exclusively for respiration.”
Darwin here seems to lean towards Lamarck. If he were right, that natural selection somehow caused the conversion, might it be through epigenetic switching prompted by conditions of existence, advantageous enough to survive as adaptation? Among others leaning towards Lamarck, Jablonka, Fox et al. (18) hold – controversially – that:
“it is quite wrong to think of the environment as just a selector of heritable variation. The environment has a dual role in evolution – it does not just select among heritable phenotypic variations, it also induces them” (my italics).
Origin’s final words reveal Darwin’s sense of wonder that “from so simple a beginning endless forms most beautiful and most wonderful have been, and are being evolved.” (14) Our growing awareness of mechanisms of epigenetics and genomic imprinting makes us all the more sensitive to the importance of keeping today’s increasingly artificial conditions of existence, as beneficial as to evolution as we possibly can.
Evolutionary concepts from the 19th century to today
David Marsh (12) well describes how Lamarck’s concept of heritable acquired characteristics, modified by Darwin, had been flatly contradicted by Weismann. Weismann held a “germ plasm theory” of sealed germ-cells, insulated against all somatoplasmic influence, ruling out heritability of acquired characteristics. Weismann’s ridiculous “disproof” of acquired characteristics – by cutting off mice-tails for 20 generations to no hereditary effect (despite centuries of docking lambs’ tails, or indeed millennia of human male circumcision)! – led to a widespread view of mutations being purely random, advantages enduring through natural selection. Such a concept of evolution that became inappropriately termed “neo-Darwinism”, many scientists even referring to it mistakenly as “Darwinism”. By the 1930s mathematical coordination of neo-Darwinism with Malthus’s principle of population growth, Mendel’s statistical approach, and human population genetics led to “the Modern Synthesis”. Setting the seal on this rigid “primacy of DNA” was the 1950s discovery of the double helix by Watson, Crick et al. reinforcing the “blue-print” paradigm. All along, caught up in the controversies were the Church, Creationism and Intelligent Design; politics, industrial revolution, and trade with religious expansionism (12). In the turbulence Darwin’s image became stripped of his wavering religious propensity, to be branded agnostic, even atheist, and his image became stripped of his profound environmentalism “Conditions of Existence”.
The opinion of David Marsh and Michael Crawford has not wavered since their publication – over a decade before the human genome was mapped – of The Driving Force: Food in Evolution and the Future, 1989, that natural selection and environmental conditions work hand in hand, as current epigenetics research is now showing. To these two authors I am indebted for their insights in Nutrition and Evolution, as their excellent book was renamed in 1995 (13).
For the controversial background I strongly recommend David E. Marsh’s The Origins of Diversity, (12) an excellent short history of evolutionary debate. It ranges from the late 18th century – Lamarck’s theory (15) – through to the human genome sequencing and current surge in epigenetics research, which is beginning to clarify precise mechanisms of evolutionary change.
I pay tribute to Michael Crawford particularly for his globally outstanding research focused on maternal–child nutrition and brain development. Crawford and Marsh are among the increasing number who stress the value of young children’s developing a taste for fish, sea foods, seaweeds and beneficial algae like spirulina and chlorella. For the sake of future generations people need to eat more aquatic foods, as the main and richest source of brain specific nutrients, docosahexaenoic and eicosapentaenoic acids (DHA & EPA) whose dietary depletion in many countries is threatening the brain (19,20,21). Covering 70% of our planet’s surface, water represents our greatest future food potential, while demise of ocean plant-life can seriously aggravate climate change. At our peril do we continue wrecking our evolutionary habitats of oceans, rivers and lakes. Their saving and enhancement, rooted in agriculture of the ocean beds, is a vital contribution to our future. Epigenetics makes even clearer the urgency of guarding aquatic habitats, if only for the sake of human health.
Many epidemiological and molecular researchers are exploring transgenerational effects, including genomic imprinting, opening windows on modifications in species. Among them are Barry Keverne (22,23,24), Marcus Pembrey (25,26,27,28), Golubovsky (29), Marilyn Monk (30,31) and Jonathan Mill (33a,33b). We seem on the verge of clarifying the relationship of heritability, yet reversibility, to more permanent changes in DNA sequence, namely, mutations. David Marsh (12) sees this as the threshold of a new paradigm. We are recognizing the molecular variations through which we are affected by our great-grandparents, and in turn affect our great-grandchildren-to-be, passing on generation by generation much of what we inherit, and a substantial part of how we live. How substantial? Despite Barry Keverne’s description (22) of genomic imprinting as coadaptive, the results of epigenetic transmission to the next generation, are still controversial – shades of Weismann?
Keverne writes (22):
“Genomic imprinting acts primarily through key regulatory genes which in turn have a cascade effect through other genes. Possible effects vary widely, for instance the mother’s food intake and weight gain; maternal fat and blood glucose; letdown of milk and post-natal pup growth. Other affects include her maternal behavior, nest-building, and placental hormones, placental blood flow and nutrient transfer, fetal growth, and early weaning and puberty onset. In these ways the placenta enables the fetus to regulate its own destiny, mainly by genomic co-adaptation affecting hormonal action on receptors in the maternal hypothalamus. The two genomes, infant and maternal, are co-adaptive for infant wellbeing and reproductive success. Offspring that have extracted ‘good’ maternal nurturing will be genetically predisposed towards good mothering” (22).
When David Barker describes the pregnant woman as reading her environment for her grandchildren, adaptively is the implication (34). Marcus Pembrey’s epidemiology indicates environmental effects in the gender-line and the trans-gender-line (25), opportunities for which are revealed in Golubovsky and Manton’s three-generational physiology of female epigenetics (29). Golubovsky and Manton indicate that establishment and maintenance of epigenetic states is a flexible and vulnerable process citing Singh et al. (35), that environmental factors (such as pollution, nutrition, and lifestyle) influence the epigenetic dynamics of the oocyte in the maternal grandmother and mother possibly causing genotype/phenotype changes in the grandchildren citing Issa (36). Boyano, Andollo et al. report mammalian male imprinting at different stages over time(37), while Grandjean and Rassoulzadegan (38) identified epigenetic inheritance mediated by RNA and micro-RNA released by sperm. They consider RNA molecules present in the spermatozoon head may be possible vectors for the hereditary transfer of epigenetic modifications.
Evidence – epidemiological and molecular
Evidence – epidemiological, transgenerational and molecularMarcus Pembrey has provocatively referred to himself as “neo-Lamarckian” – in apparent support of adaptive evolution. He and Marilyn Monk, both of London’s Institute of Child Health, are among those describing transgenerational transmission of environmentally induced epigenetic change. They include epidemiological and molecular evidence of parent to child transmission (25,26,39,30) with particular susceptibility during reproduction and development.
Epigenetics has qualified Mendelian theory in that randomization studies need not only to assume a random distribution of alleles in the offspring, but also a random distribution of epigenetic changes at conception, in order for the core assumptions of the Mendelian randomization methodology to remain valid (32).
Although their effects were recognized early in the 20th century, epigenetic mechanisms were scarcely being revealed until its last decade. Then in 2001 the shock finding of the Human Genome Project completion – with less than a quarter the number of genes expected – turned minds to explore gene-expression to explain the immense variations in human beings, and their fine-tuning to their environment. The result is a reasonable explanation of two mysteries – (a) how environment can stamp its mark on an organism’s phenotype and (b) how, within an organism, the position of each cell might induce its specific role-development – both by this single type of process. Yet neat as this explanation may be, it is in fact a complex range of processes, as will be summarized.
Inheritance and genomic imprintingGenomic imprinting tends to stabilize transmission from parents to child with apparently rare changes to imprints. Imprints are usually sustained in somatic cells through life – see under “Human reproduction, Physiological stages of imprinting”, below (40). Flexibility comes through environmentally caused epigenetic changes affecting only the individual, but including the fetus or infant indirectly through the mother. Highly complex processes are involved in various reproductive processes: oogenesis and spermatogenesis, fertilization and early embryonic development (2). The most complex and seemingly most significant stage is periconceptional, from just before conception up to gastrulation – the beginning of the morula’s nutritional opening of what is to become the alimentary canal (41). Notably some specific gene-switch settings are inherited alleles – specifically as active or inactive. This process of genomic imprinting is accredited by Keverne (22) with the value of co-adaptive genetic behavior: paternal, maternal, embryonic and placental. In contrast, genomic imprinting is open to danger. Being mono-allelic, an imprinted gene lacks the safeguard of an alternative copy of the gene similarly marked. Should one be flawed, it can result in a genetic disorder, for instance Prader–Willi or Angelman syndrome. Following gastrulation there is growing evidence of epigenetic effects from nutrition in gestation and infancy, (42,43,26) and more strongly from stress, cortisol (7,8,44,45,46,47,48,49).
Tell-tale rapid changesSetting innovations in their evolutionary context provides an impression of the interplay between evolving organisms and evolving mechanisms. Significantly we recognize some of our conditions of existence to be changing now so fast and so adversely as to introduce some traits into our evolution that some hold to be seriously degenerative, such as the sudden escalation in obesity and diabetes, cardiovascular and mental disorders. Technologies that contribute to this could also provide insights and tools to contend with the increasing complexities. Epigenetics highlights the need for farsightedness in matters of human health. To give some substance to this view, Crawford and Sinclair (50) prophesied in 1972 that whilst heart disease and cancer headed the burden of ill-health in the 20th century, mental ill health would replace them in the 21st century. A terrible confirmation of their prophecy is its fulfillment. In the UK the annual £77 billion cost of brain disorders is more than cancer and heart disease combined (51). From the U.S. Department of Health and Human Services we read, “The cost of treating mental disorders rose sharply between 1996 and 2006, from $35 billion (in 2006 dollars) to almost $58 billion? 27 million people, were using antidepressants in 2005, compared to 5.84%, or 13.3 million people, in 1996” (52). For Europe, “? the true economic cost of disorders of the brain is substantially higher than our estimate of 386 billion Euros, perhaps in the range 500–700 billion Euros” (53). The gene sequence has not changed (54): conditions of existence are now constantly changing, and affecting epigenetic settings.
Time perspective – the Big Bang– beginning of evolution – into the future
Inorganic matter to cellular life
The beginning of life and accelerating evolutionary processes need to be seen in perspective, against the Big Bang some 15 billion years ago (bya), the earth’s formation some 4.5 bya and assembly of elements into molecules. In the submarine volcanic heat fatty acid molecules may have formed on minerals, then conglomerated, drawn together by their oily end into spheres, their water-soluble ends outward in the ocean. Possibly RNA was established by this stage. A likely date-scale suggests amino-acids made feasible the protocell, “prokaryote”, some 3.8 bya, then DNA and microbes by 3.5 bya, with powerfully photosynthetic cyanobacteria by 3 bya. By 2.8 bya cell nuclei formed and eukaryotic cells, the basis of today’s immense variety of life, with cell memory, light-sensitivity and photosynthesis. By 2.5 bya the ocean may have been rich in omega-3s, docosahexaenoic acid (DHA) and eicosapentaenoic (EPA), significant particularly to the blue-green algae and phytoplankton, with the new process of photosynthesis, binding carbon. So successful were they that they threatened themselves with excess of their own oxygen, until rescued by the mitochondria at 2.3 bya. Burning oxygen back to carbon dioxide were the mitochondria, symbiotically with the blue-greens and phytoplankton on the one hand, and on the other hand not just energizing themselves but becoming the new range of oxygenated life-forms. This vast evolutionary potential remained poised on the threshold, inhibited by the deepest of all ice-ages, for some billion and a half years. Then, 670 million years ago (mya) a new warm climate brought on a burgeoning variety of new life, the Cambrian Explosion, evidence of which was first unearthed among the ancient rocks of Wales (Cambria).
Further key transitions in evolution with likely changes in epigenetic processes
Million years ago (mya):
670 from single to multi-cell life
540 to shell-bearing life
500 vertebrate life
400 amphibians and vitamins for land-life
180 marsupials and mammals diverge
130 flowering plants, seeds with omega-6s
50 sea mammals with omega-3s
2 Homo’s brain enlargement with omega-3 docosahexaenoic acid (DHA).
Once this oxygenated life became multi-cellular, it sprang to highest complexity, shell-bearing invertebrates, vertebrates and brain-powered life. Within 200 million years legs had appeared and amphibians. Vitamins, in evidence by 350 mya, seemed a requirement for land-life metabolism. A significant benefit featured in the transition between aquatic and terrestrial life, from a diet combining omega-3s with omega-6s, the very prescription for both the placenta and for brain enhancement. So the leap forward: reptiles 360 mya, mammals 210 mya, and Homo habilis 2 mya. This perspective spanning from the Big Bang to the present shows the long time-scale and widely varying pace of evolution with the sense of its continuity into the future. This is important in grasping the rapidity of a sequence of civilizational changes. One after another in a mere 400 generations, beginning with the hunter-gatherer, have fallen such impacts as agriculture, industrialization, intensification of farming, high-tech medicine, food manufacture, and marketing; and now the challenges of pandemic diseases, climate change and ecological depletion, are calling for urgent ocean-bed agriculture. Now our myriad artificial environmental interventions come at a stage when our human sensitivity has never been higher in terms of epigenetics, genomic imprinting included. Through this young field of study can we divine any moments in this lay-out of evolution during which novel mechanisms were achieved?
Although we can tell many ways that environment affects cells and organisms, and how it does so, we can largely only speculate on when and how the changes came about to accumulate to the complex systems now regulating life. The most likely indications will come from the transitional stages in evolution, found by comparing fossil records across transitions with epigenetics of the descendants we know today.
Mechanisms emerging in evolution
Subcellular, cellular and gene-related
Switching mechanisms in evolution
There are many mechanisms affecting gene settings whose activity or silence depends on spatial structure of molecules. A small proportion of these have been worked out. Discerning which species first achieved a mechanism, and at what stage in its evolution, reveals some of the major moments in evolution at which changes in organisms and mechanism took place. Some landmarks:
1. Chemical activation and inactivation (silencing) is not confined to genes but can operate in nucleosomes around which the chromosomes wrap themselves, affecting their spatial positioning and so the organism’s life.
2. Even protocells and single-cell organisms – some 3.8 to 2.7 bya – needed components with some kind of marking for their self-arrangement and replication. With the assembly of DNA at 3.5 bya, and cell-memory at 2.8 bya, epigenetics would presumably have begun. Some such mechanisms might have been operating in RNA, which could possibly have preceded DNA. RNA is the less stable. It not only has a part in transcribing proteins but in transcribing back to the DNA as influenced by environment.
3. By 2.7 bya eukaryotes existed, with responses to environmental changes becoming increasingly elaborate. From this divergence of eukaryotes from prokaryotes, at 2.7 bya, springs sexual reproduction (55) with increasingly complex mechanisms.
4. In multi-cellular organisms, by 670 mya, each stem-cell needs markings not only for its internal components but to differentiate appropriately for its position. Cell-to-cell communication may help in this process (56). Markings basically remain according to initial settings in the embryo except as changed by immediate environmental conditions.
Two common mechanisms – DNA methylation and histone acetylation
Two of the commonest mechanisms, and best understood, are DNA methylation and histone acetylation. Switching genes off is by attachment of methyl groups to cytosines, several in a cluster, or by detaching acetyl groups from lysines on histones. Often the two processes work together, tightening the way that DNA is coiled around the “nucleosomes” (spheres of histone protein), spatially changing the shape and physical forces of the DNA system. Histones also may be methylated, which may either turn a gene on (H3K4me2,3), or off (H3K9me2,3). Silencing genetic expression of is mostly through switching off controlling genes upstream. Changing gene expression substantially affects transcription. Some yeast cells, for example, can abruptly change from single sex to a capacity for switching between two sexual types. Most epigenetic changes are made through regulatory genes, having a cascade affect on many genes.
Yet methylation and acetylation are just the two best known mechanisms, and there are many others at play. Zuckerkandl, for instance, describes how “junk DNA” along the chromosome is probably influencing the extent to which methylation is happening. Influences such as this may be affecting how lasting the epigenetic change may become, and whether it may cause a change in DNA sequence, a mutation (57).
Relationship in evolution between genetic changes and epigenetic
Mutations, irreversible, are almost invariably passed down to half the children. Epigenetic settings, reversible, are mostly erased during early embryo implantation: generally imprints only are re-established. A key question is the relationship between epigenetic changes and mutations. Current speculation is that changes in gene sequence – mutations – correlate with lasting composite epigenetic changes (61).
The power in evolution of various mechanisms of epigenetic transmission is emphasized by Jablonka and Lamb (18), who write of the combinations “?of different active/inactive loops, cellular architectures, chromatin marks, and RNA-mediated silencing patterns.” The amount of cell variation, they say, is vast and the evolutionary potential of inheritance systems therefore considerable. They draw the general conclusions that when conditions change, epigenetic events can increase the rate of adaptive evolution, by activating silent genes and through heritable variants. They also quote Belyaev’s (59) work in Russian with the silver morph of the red fox. He took two groups of silver foxes, farm-raised one and domesticated the other, observing the differences between each group and those remaining in the wild. Even over 10 generations remarkable differences emerged in coloring, behavior, leading him to conclude that “induced heritable epigenetic variations play an integral role in adaptive evolution.” Although the epigenetic functional differences were not analyzed, Belyaev detected 40 gene differences between domesticated and farm-raised, and between them and wild foxes 2700 different genes (60).
Belyaev’s demonstration how few generations of silver foxes it takes to induce heritable adaptive changes, combine with such human transgenerational effects such as Pembrey’s Swedish study (see section below on “Transgenerational effects”) keep open the search for mechanisms that can, so quickly, and often enough favorably, select from alternative settings, with those lasting composite epigenetic changes that correlate with mutations. Rando and Verstrepen (61) have attended to time-scales of variations and suggest that some organisms have evolved mechanisms with variability that relates to the variability of selective pressure encountered. The search for non-teleological adaptive mechanism continues.
Single cell to amphibians
Early processes in protocells, nuclei and memory
Components and cells, and thereby organisms, are affected by factors including: component position and shaping; memory-responses; neighboring components; gravity and pressure; and environment – chemical, electromagnetic, and radiation – cosmic, geological, artificial. Some of these environmental forces can induce mutations. Since cells, or even organelles, require components to organize themselves appropriately to their position, we can reasonably assume markers or signals to be involved at a very basic level. We are at least clear that DNA strands have markers making fine distinctions (62). Memory can lie latent yet ready to be reactivated (63). DNA methylation can be altered by “activation-induced (cytidine) deaminase” (AID), an enzyme involved in the formation of the immune system, leaving cells with inaccurate memory. AID initiates immunoglobulin class switch recombination and somatic hypermutation by producing uridine:guanadine mismatches in DNA, which can also induce DNA damage including double-stranded breaks and chromosome translocations. Strict regulation of AID is vital for genomic stability (64). Wolf Reik proposed that methyl-cytosine can be changed to thymine by deamination with AID, being repaired to normal cytosine (65). Nat Heintz showed that methyl-cytosine can be converted to hydroxymethyl-cytosine by other enzymes and is similarly repaired (66). See also AID in the paragraph after next, “Invertebrate to vertebrate transition”.
Bacteria and mitochondriaBacteria make widespread use of DNA methylation, following replication, for epigenetic control of DNA-protein interactions. Bacteria methylate adenine in DNA, rather than cytosine, as an epigenetic signal (67). Mitochondria evolved possibly within bacteria at the same time as the eukaryotic cell nucleus (68,69). Yet they seem to have emerged independently and entered eukaryotes, and symbiotically have provided signaling, regulating nuclear gene expression, (70) and oxygen-powered energy (13). Mitochondria DNA has a mutation rate 10 times that of eukaryotes. Somehow, with their signaling role, this has potential to speed up mutations to rates more closely compatible with the recognized pace of evolution (71). The mitochondria are rewarded with access to plentiful oxygen. The radically diverse trends in mitochondrial genome evolution, recognized in different phylogenetic groups, has allowed pinpointing of specific protist relatives of multicellular lineages – animals, plants, and fungi. This research revealed “unique and fascinating aspects of mitochondrial gene expression, highlighting the mitochondrion as an evolutionary playground par excellence” (72).
Invertebrate to vertebrate transition
DNA methylation is a mark associated with gene regulation and cell memory, silencing of transposable elements, genomic imprinting, and repression of spurious transcription of duplicated sequences. These roles have varied widely during animal evolution. DNA-methylation machinery includes three groups of enzymes (Dnmt methyltransferase), and five binding proteins (Mbd methyl-DNA).
Albalat (73) has identified changes in the presence of these Dnmt and Mbd gene families at the juncture between invertebrates and vertebrates (in the cephalochordate amphioxus, Branchiostoma floridae), a group closest to vertebrates. Whereas three major groups of Dnmt enzymes were found in the invertebrates, in the vertebrates only two Mbd members were found. Although during the invertebrate-vertebrate transition, methyltransferases were little changed, new Mbd proteins arose, which perhaps minimized certain collateral effects associated with the major genomic changes that occurred.
Between fish and tetrapods appeared class switch recombination, the last of the lymphocyte-specific DNA modification reactions to appear in the evolution of the adaptive immune system. Class switching is initiated by activation-induced cytidine deaminase (AID), which is also required for somatic hypermutation. Fish AID differs from orthologs found in tetrapods in several respects, including its catalytic domain and carboxy-terminal region, both of which are essential for the switching reaction. Fish AID was found to catalyze class switch recombination in mammalian B cells, and therefore had the potential to catalyze this reaction before the teleost and tetrapod lineages diverged (74). See AID in “Early processes”, above.
Amphibians – reversion potential of somatic cells to stem cellsSalamander somatic cells have been remodeled to stem cell state, clearly a “reverse” epigenetics change. The levels of signal intensity in differentiated cells that are then treated, resemble those detected in embryonic stem cells, which are unaffected by these extracts. Selectively somatic cells exposed to oocyte extracts undergo demethylation (75). Plants, fish and animals, that abandoned the sea for the land and inland waters, lacked a wide range of nutrients, iodine, and other elements, also marine antioxidants, prompting improved production of various antioxidants which became essential vitamins to life on land.
Mammals – transitions to placenta, live birth and genomic imprinting
Genomic imprinting marks a gene active or inactive mono-allelically according to its being the maternal or paternal allele. It is mainly evident in some flowering plants and in placental mammals. Origins of mammalian genomic imprinting are emerging from studies of two transitions: (a) from egg-laying “prototherian” mammals such as the platypus, with only a short-lived placenta, to fully placental “therian” marsupials such as the kangaroo; (b) from therians on to fully placental “eutherian” mammals. Since even egg-laying prototherians have a short-lived placenta, imprinting appears to correlate with giving live birth (viviparity) rather than with placentation. Marsupial live-birth follows a short gestation supported by a fully functional placenta (76,77).Mammalian acquisition of genomic imprinting – (from prototherian to therian to eutherian)The acquisition and evolution of genomic imprinting is among the most fundamental genetic questions. Genomic imprinting is an epigenetic phenomenon that regulates many aspects of growth and development. Apparently absent from the egg-laying prototherian mammals such as the platypus, genomic imprinting is widespread in (therian) marsupials, such as the kangaroo, as well as more advanced (eutherian) mammals. According to favored hypotheses, genomic imprinting evolved within the cell, benefitting it by silencing foreign DNA elements entering the genome, and by balancing maternal and fetal nutrient supply (77,78).
Pask, Papenfuss et al. (78) showed that the platypus has significantly fewer repeats of certain classes in the regions of the genome that have become imprinted in therian mammals. They conclude that the accumulation of repeats in therian imprinted genes and gene clusters, especially long terminal repeats and DNA elements, may have been a driving force in the development of mammalian genomic imprinting. All orthologs of eutherian imprinted genes examined have a conserved expression in the marsupial placenta regardless of their imprint status.
In eutherian mammals the most common mechanisms controlling genomic imprinting are “differentially methylated regions” (DMRs), whereas in the marsupial the mechanism used to silence the equivalent genes appears to be histone modification.
“At least three genes in marsupials have DMRs: H19, IGF2 and PEG10. PEG10 is particularly interesting as it is derived from a retrotransposon [a DNA sequence that can move its position within the genome], providing the first direct evidence that retrotransposon insertion can drive the evolution of an imprinted region and of a DMR in mammals. The insertion occurred after the prototherian-therian mammal divergence, suggesting that there may have been strong selection for the retention of imprinted regions that arose during the evolution of placentation” (77).
Non-coding RNAs in mammalian acquisition of genomic imprintingMost imprinted genes, located in clusters and regulated by imprinting control regions, included non-coding RNAs. Some of these non-coding RNA-expressions were changed dramatically at this stage, and also many novel non-coding RNAs were added. A study of imprinted small nucleolar RNA genes from 15 vertebrates “suggests that the origination of imprinted snoRNAs occurred after the divergence between eutherians and marsupials.” Subsequently rapid expansion led to the fixation of major gene families in the eutherian ancestor, and then the radiation of modern placental mammals. The non-coding RNAs’ major roles during the acquisition of genomic imprinting in mammals seem to be the regulation of imprinting silence, and mediation of the chromatins’ epigenetic modification (76).
Imprinting differences in the mammalian embryo and placenta
The maternal genome in the zygote is highly methylated in both its DNA and its histones, its imprinted genes mostly having maternal germline methylation imprints. The paternal genome is rapidly remodeled by protamine removal, addition of acetylated histones, and rapid demethylation of DNA before replication. A minority of imprinted genes are silent, having paternal germline methylation imprints. Methylation and chromatin reprogramming continues during cleavage divisions. At the blastocyst stage, DNA and histone methylation increases dramatically.
This may set up major epigenetic differences between embryonic and extraembryonic tissues, placenta included. X-chromosome inactivation is involved and perhaps imprinting. Maintaining asymmetry appears important for development. In cloned embryos asymmetry is lost, most having developmental defects, particularly an imbalance between extraembryonic and embryonic tissue development (79).
New sexual and parenting behavioral imprints in hominids
Two major developments have affected mammalian sex differences in behavior: the placenta’s hormonal effects on the maternal brain, especially in small-brained rodents; and the brain’s massive expansion in primates, especially hominids. In both developments genomic imprinting has been significant. “Most of the imprinted genes investigated to date are expressed [mono-allelicly] in the placenta and a subset are expressed in both placenta and hypothalamus.” Recent knock-out studies suggest the coadaptive effect of imprinting may have been significant in imprinting’s evolution, rather than parental conflict as commonly held, contributing to coadaptation between male and female and offspring. Evidence supports a coadaptive evolution of placenta and hypothalamus, particularly in neurohormonal regulation of maternalism. The neocortex and other parts of the brain which have expanded are undoubtedly under the influence of imprinted genes. In small-brained mammals, a female’s short estrus demands greater olfactory powers from males, compensated by a male accessory olfactory system. Evidently the same imprinted gene that regulates mammalian maternal care and offspring development also regulates male olfaction and sexual behavior. In hominids, humans particularly, differences in sexual behavior owe much to social structure and strategies of intelligence. Social learning has become as important as hormones in epigenetic effects on brain development (23,24).
Stability of the original chromatin strand in differentiating cells
From problems including cancer and atheromatous plaque increase with aging, we learn about protection of chromatin strands. In healthy cell division the stem cells retain the original chromatin strands while the differentiated daughter cell receives new chromatin strands, which are more susceptible to error in self-repair. Since a differentiated cell has a limited life before dying and being replaced, there is little chance of a problem. Occasionally, however, the differentiating daughter cell receives the original strands, leaving the stem cell with the newer strands in which any fault will continue to be replicated. Epidemiological time-patterns indicate a mechanism that almost always prevents “a final step” of mutation to cancer. The final step could be when a rare fully mutant cancerous stem cell produces a daughter cell that is freed from some nongenetic imperative to differentiate and die. This appears to be a mechanism, like the one that induces appropriate cell differentiation, which marks the strands as original or new so that persistent errors are effectively prevented (52).
According to D. Simmons, “although epigenetic changes do not alter the sequence of DNA, they can cause mutations. About half of the genes that cause familial or inherited forms of cancer are turned off by methylation. Most of these genes normally suppress tumor formation and help repair DNA, including O6-methylguanine-DNA methyltransferase (MGMT) . . For example, hypermethylation of the promoter of MGMT causes the number of G-to-A mutations to increase”.
Unlike DNA sequence mutations, which are irreversible, many diseases such as cancer involve epigenetic changes. Being reversible, these could be responsive to epigenetic treatment. The most popular of these treatments aim to alter either DNA methylation or histone acetylation (81).
Gender effects as well as sex-chromosome effects in health and disease
The vast majority of common diseases, including atherosclerosis, diabetes, osteoporosis, asthma, neuropsychological and autoimmune diseases, which often take root in early development, display some degree of sex bias, very marked in some cases. This bias could be explained by the role of sex chromosomes, the various regulatory pathways affecting sexual development of most organs and the continuing fluctuating impact of sex hormones.
In a gender-related manner, environmental factors such as social behavior, nutrition or chemical compounds can influence these flexible marks during particular developmental stages and subsequent changes in life. Each developmental process may be more sensitive for one gender or the other during specific environmental challenges, particularly developmental programming and gametogenesis, but also throughout the individual’s life as influenced by sex steroid hormones and/or sex chromosomes. An unfavorable programming could thus lead to defects and susceptibilities to diseases differing between males and females. Recent studies suggest that such programming can be sex-specifically transmitted to subsequent generations leading to transgenerational effects. Gabory et al.’s review highlights the importance of studying both sexes in epidemiological protocols or dietary interventions whether in humans or in experimental animal models (82).
In the mouse gender-dependent genomic imprinting effects, not related to sex-chromosome effects, have been demonstrated. 13 loci on 11 chromosomes showed significant differences between the genders in imprinting effects. Most loci showed imprinting effects in only one sex, with eight imprinted effects found in males and six in females, but one locus showed sex-dependent imprinting effects in both sexes for different traits (83). The degree of imprinting is often tissue-specific or developmental stage-specific, or both. In some diseases, cancer included, it may be altered. Some 1% of genes may be imprinted and the balance between alleles can vary from 100% one-way to 50–50 (84). Wang et al. found in neonatal mouse brains that both known imprinted genes and novel genes were all close to differentially methylated regions (DMRs).
Human reproduction – health of future generations
Origins of health and disease – primacy of epigenetics
Epigenetics could contribute importantly to lifelong prevention of common chronic health conditions. Focus of The International Society for Developmental Origins of Health and Disease (DOHaD) (6) is on the earliest stages of human development. The Society’s 3rd International Congress, 2005, added new perspectives, including developmental plasticity, influences of social hierarchies, effects of prematurity, and populations in transition. Emerging areas of science included:
- Infant weight gain and prediction of adult obesity, diabetes, and cardiovascular disease.
- The era of epidemic obesity – the over-nourished fetus and growth retarded one.
- Environmental toxins’ broad range of long-lasting effects on the developing human.
The Society recognized that epigenetic mechanisms could unite several strands of human and animal observations. They could explain, for instance, how genetically identical individuals, even raised in similar postnatal environments, can nonetheless develop widely differing phenotypes (85,86,87,88). Improving the individual’s environment during development may be as important as any other public health effort to enhance population health world wide.
Nathanielsz uses of animal models to evaluate specific exposures such as nutrient restriction, overfeeding during pregnancy, maternal stress, and exogenously administered substances such as glucocorticoids on developmental programming, revealing effects of hypertension, diabetes, obesity, and altered pituitary-adrenal function in offspring in later life. Although for example, the fetus responds to challenges such as hypoxia and nutrient restriction in ways that help to ensure its survival, this “developmental plasticity” may have long-term consequences that may not be beneficial in adult life (89).
Recent findingsDuring development, there are critical periods of vulnerability to suboptimal conditions when programming may permanently modify disease susceptibility. Programming involves structural changes in important organs; altered cell number, imbalance in distribution of different cell types within the organ, and altered blood supply or receptor numbers (90).
Transgenerational sex-specific links were evident in a study of Swedish cohorts by Pembrey, Bygren et al. (25). The paternal grandfather’s food supply was associated with the mortality risk ratio of grandsons only, and the paternal grandmother’s with the granddaughters’ only. The effects followed exposure during the slow growth period (both grandparents), or fetal/infant life (grandmothers), but not during either grandparent’s puberty. These sex-specific, male-line effects in humans suggest mediation by the sex chromosomes, X and Y. The same study showed early paternal smoking, before 11 years old, to be associated with greater body mass index (BMI) at 9 years in sons, but not daughters. A subsequent study by Kaati, Bygren et al. (26) confirmed these effects. It also showed that early social circumstances influenced longevity for sons. The main influence on longevity was transgenerational responses to ancestors’ nutrition. But less expected was Pembrey’s study (27) showing scarcity of food in the grandfather’s slow growth period to be associated with a significantly extended survival of his grandchildren for many years, whilst food abundance was associated with a greatly shortened life span of the grandchildren. Even more surprising is that overall they show that cardiovascular mortality was reduced with poor availability of food in the father’s slow growth period, but also with good availability in the mother’s slow growth period? “hinting at some ‘see-saw’ effect down the generations”.
What is known about these mechanisms? Hung, Farooqi et al. including Pembrey (91) studied effects of a particular variant gene, the “small heterodimer partner” (SHP), which has repressive effects hormonally and transcriptively. They concluded that although mutations in SHP are not a common cause of severe human obesity, genetic variation in the SHP locus may influence birth weight and have effects on body-mass index (BMI), possibly through effects on insulin secretion. Even in 1996 Pembrey (28) was speculating on the beneficial potential of such knowledge. Poised between transcriptionally active and silent states, imprinted genes seem good candidates for the evolution of transgenerational adaption systems, where coordinated changes in gene expression over the generations are a selective advantage.
Physiological stages of imprinting processes
Clearly the process of imprinting is complicated and Barry Keverne does not claim to know the full story yet, but I relay here the 4-stage cycle with two notes that he has kindly set out (40):
1. Epigenetic reprogramming events occur in the primordial germ cells at embryonic day 8. This results in a global loss of methylation.
2. Following this erasure there is a resetting of the epigenome in the germline – this may account for those instances of transgenerational inheritance of epimutations.
3. Later in oogenesis and spermatogenesis, “de novo” methylation occurs in a sex-specific manner for imprinted genes.
4. There is a further wave of genome-wide reprogramming events immediately after fertilization, but imprinted genes are thought to be protected from demethylation in the zygote.
(a) The general rule is that the imprint is maintained throughout development, so all somatic cells that express this imprinted gene do so in a haploid dominant manner according to parent of origin, i.e. the memory is sustained through mitotic divisions.
(b) This imprint never switches in somatic cells, only in germline cells following reprogramming. For the most part, imprints retain their mono-allelic stability, except in tumors. It has also been shown that handling of fertilized embryos (e.g. in-vitro fertilization/embryo transfer) can also influence imprints.
This sequence makes evident:
- Some reasons for high vulnerability of the fertilization/early-embryonic phase – in 1, 4, and (a).
- A likely stage at which occasional epimutations may be transmitted – in 2.The setting of sex-specific imprinting – in 3.
- The way in which imprint memory is sustained for transmission transgenerationally – in (b).
Continuing evolutionary impacts on health and disease
Subtle environmental changes throughout early development affect later health and disease. Genetic evolutionary background contributes significantly to our susceptibility to perinatal imprinting. Epigenetic modulation, in reaction to a given environment, results in functional adaptation of the genomic response, more plastic than the genome sequence.
“Evolutionally acquired genomic susceptibilities, and environmentally induced epigenomic modulations occurring early in life, impact on later development of human diseases” (92).
Such evidence as amply supplied by J. Tremblay and P. Hamet (92) provides molecular understanding that might explain some of the extensive evidence gathered in The Unborn Child (21).
For half a billion years there has been no change in our inherited genetic mechanism for human blood clotting. Genetically it remains the same as the puffer-fish, reports hematology professor Edward Tuddenham. Yet in a mere 150 years our cardiovascular problems include a huge thrombosis epidemic with high mortality. The cause is changes in blood-clotting due to smoking and modern diet. “The cure must lie in returning our diet towards its premodern state” (54). Epigenetic implication is supported by studies including that by P. Sharma, R.D. Senthilkumar et al. (93).
Studies of genes in relation to violence so far have largely concerned the MAOA gene, which in children, only after maltreatment, correlates with nine times the risk of violent behavior (94). This seems ripe for epigenetic study. Professor Bruce Lipton perceives a process of “survival of the most loving.” Scant nurture or other severe conditions, he writes, will program the new being for anxiety about survival: generous nurture will program a child for trust, love and creativity, for reproducing and nurturing (95).
Bridging between cytology and psychiatry between 1954 and 1982 was a scientist, Dr Frank Lake (7,8,9) After 13 years of studying cell memory, Lake qualified in psychiatry. At first using lysergic acid diethylamide 25 (LSD25), but then finding therapy more effective through current feelings and memory evoked without any drug, he found patients were accessing early memories and also, through physical movement, very early cell memory. Lake’s discoveries were contemporary with Arthur Janov’s (96,97) yet independent, and had striking similarities, particularly in re-experience of birth. Since then this experience has become common to those involved in pre- and perinatal psychotherapy. Lake’s insights have been incorporated in William Emerson’s renowned work (98). Lasting psychological effects of human gestation and birth have been set in an evolutionary context by Ludwig Janus (99). Lake went on to train teams of psychotherapists. Patients and therapists, including medical people, became convinced that powerful body movements were expressing cell memory from early somatic or cellular memory from as far back as conception. I am not implying embryonic consciousness, nor that such early events were “remembered”. Karl Pribram held that a short-term memory could resonate through the brain’s stored holograms until an association is triggered in long-term memory. According to Lake (100, 3):
“The holograms of cellular memory are still broadcasting from infinitesimally small, but collectively audible transmitting stations. These minute radio stations belong to successive periods of development, from conception to implantation and the developmental stages of pregnancy. It seems they are still transmitting and it is possible to tune into them.”
Among thousands of the Lake teams’ patients, for many the periconceptional and embryonic stages seem to have had the greatest permanent effects on the person’s later feelings and behavior. Inevitably controversial, those of us with these experiences find them hard to dissociate from earliest cell memory. Evidence of epigenetic effects at stages round the lifecycle help to make such findings more intelligible, and may well cast new light on early life plasticity. Extensive records of pre- and perinatal psychotherapists witness to the impacts of early life circumstances, which help to unravel the effects of cultural ways on the emotions and mind-set of generations (101,102).
Mental disorders and heritability, and psychotic drug effects
Alarming epigenetic evidence relating to mental disorders includes long-term antipsychotic drug use and transgenerational effects. These further strengthen the impression of our continuing evolutionary state. J. Mill, A. Petronis et al. (33a) describe correlations of epigenetic misregulation with various non-Mendelian features of schizophrenia and bipolar disorder, including several affecting brain development, neurotransmission, and other processes. Epigenetic disruption was evident in mitochondrial function, brain development, and stress response.
Like the DNA sequence, the epigenetic profile of somatic cells is mitotically inherited, but unlike the DNA sequence, the signals are dynamic. The epigenetic status of the genome is tissue-specific, developmentally regulated, and influenced by both environmental and random factors. Genes must therefore be both in the right sequence and also expressed in the appropriate amount, at the correct time of the cell cycle, and in the correct compartment of the nucleus. During gametogenesis some epigenetic signals, rather than being erased and reset, can be transmitted meiotically across generations (103,104). For both brain and germline DNA, evidence suggested that the epigenome is regionalized relating to distinct physical regions and/or functional pathways. Comparing the affected group to the control group:
(a) The number of interconnections between genomic regions is higher, resulting in more interference between regions in both brain and germline DNA, and therefore very possibly between specific functional tasks (105).
(b) In the major-psychosis samples, these regions’ greater activation (lower degree of DNA-methylation), indicates some degree of systemic epigenetic dysfunction associated with major psychosis.
“Our data are consistent with the epigenetic theory of major psychosis and suggest that DNA-methylation changes are important to the etiology of schizophrenia and bipolar disorder” (33a).
Mill and Petronis also propose that understanding the epigenetic processes involved in linking specific environmental pathogens to an increased risk for ADHD may offer new possibilities for preventative and therapeutic intervention (33b).
Safeguarding human reproductive health with epigenetics understanding
For decades findings in nutrition and psychotherapy have shown the impact of periconceptional as well as pre- and perinatal circumstances on development. In the authoritative words of E.L. Ford-Jones et al:
“Diseases of modernism, rather than infectious diseases and chronic medical conditions, increasingly cause childhood morbidity and mortality. Thus, the goal of enhancing life outcomes for all children has become imperative? The new neuroscience of experience-based brain and biological development has caught up with the social epidemiology literature. It is now known from both domains that a child’s poor developmental and health outcomes are a product of early and ongoing socioeconomic and psychological experiences. In the era of epigenetics, it is now understood that both nature and nurture control the genome? A challenge is to connect the traditional population health approach with traditional primary care responsibilities. New and enhanced collaborative interdisciplinary networks with, for example, public health, primary care, community resources, education and justice systems are required” (106)...
...required to benefit not merely our health but our continuing evolution.
Significance of evolutionary differences to assisted reproductive technology
Since imprinting is related to various processes in reproduction, it is naturally liable to impacts from assisted reproduction technologies (ARTs), especially during embryo and trophoblast development, as ARTs are used during these periods.
L. Wilkins-Haug et al. found that induced ovulation, and oocytes with potentially less stable imprints, may contribute to the higher rate of the maternal imprint disorders. Paternal imprinting abnormalities in oligospermatic men may indicate that subfertility itself is associated with epimutations – low sperm quality does correlate with low sperm count (2). Higher rates of adverse outcomes that follow ARTs, such as growth restriction, may be found related to placental epimutations (107,108).
The association, particularly in assisted conception, of disease and genesis of tumors with perturbed imprinting means that monitoring for normal imprinted gene expression in human embryos is critical. Monk and Salpekar showed mono-allelic expression to be tissue-specific and time of onset to vary between different imprinted genes. Three of six genes analyzed were clearly expressed in human preimplantation embryos, and the expression of one, SNRPN, was mono-allelic from the paternal allele. This gene was also mono-allelically expressed in mouse preimplantation embryos, being “correlated with differential methylation of Xist promoter sites in egg and sperm, and specific binding of a protein only to the methylated maternal (egg) allele.” But in human preimplantation embryos, “unlike the mouse, XIST is expressed from both parental alleles.”
Monk and Salpekar’s studies highlight not only a significant step in the evolution of an epigenetic mechanism, but also the criticality of checks that animal research findings are entirely appropriate to techniques in human medicine (31).
Darwin’s final statement in Origin of the Species (14) is: “There is grandeur in this view of life?that?from so simple a beginning endless forms most beautiful and most wonderful have been, and are being evolved.” This ties in with Chapter VI’s concluding climactic paragraph “?that all organic beings have been formed on two great laws – Unity of Type, and the Conditions of Existence, [which is] the higher law?”. The term “Conditions of Existence” Darwin here introduces weightily as that of “the illustrious Cuvier”. Although using it three times in this paragraph out of only four in the main text, he does not distinguish it from his own term “conditions of life” which, in his main text, he synonymously uses no less than 118 times.
Darwin did try to explain how conditions could affect change in organisms and their heritability, offering the hypothesis of “pangenesis” (109), but it failed to hold back the general acceptance that changes were random and subject to the slow work of natural selection. Today’s awareness of mechanisms of epigenetics and genomic imprinting vindicate Darwin’s search, explaining even the power of changing conditions on organisms that we are forcibly witnessing in our lifetime. Just as very few generations of changed conditions were needed for changes in Belyaev’s silver foxes, very few generations have been needed to bring human heart disease from a rarity to top killer over the last century. Now brain disorders are overtaking the cost of all other burdens of ill health at least in Europe and spreading globally (51,52). The need is urgent to address the “Conditions of Existence” law of Cuvier and Darwin, by which most serious epigenetic impacts on Homo sapiens are threatening subsequent generations.
Epigenetics could be our most powerful technological insight against the current crises, microscopically highlighting the dependence of human health on the biosphere. The cradle of biosystems has been the estuarial and inshore beds of photosynthetic systems, basic to the marine food chain, whose demise threatens both collapse of fisheries and aggravated climate change. Of the Philippines coral reefs, only 5% remains healthy. The fight is on to restore their thousands of island shores (110).
How can we reverse the drastic trend in brain and other disorders? Draconian steps have necessarily begun, to cut the industrial pollution destroying our biosystem, and to build systems of marine agriculture, include filtering of pollution with seaweed and shellfish, as in Sungo Bay, China since 1991. Such measures are essential if only to meet the human brain’s prime need for the marine source of omega-3 oil docosahexaenoic acid and other essential nutrients, that is greatest in gestation and brain development (51,52). Healing fertile marine regions is basic to restoring human brain health in today’s mental health pandemic.
“The concept of fetal programming is an area that is now under rigorous investigation in many laboratories throughout the world. We need to engender a fascination in all segments of society, not just pregnant women, about life in the womb. Conclusion: Everyone needs to understand that improving the condition of the fetus will have personal, social and economic benefits. The time has come to realize that, in a sense, it is not just women who are pregnant but it is the family and the whole of society” (111).
Our growing awareness of mechanisms of epigenetics and genomic imprinting should raise our sensitivity to the importance of keeping today’s increasingly artificial conditions of existence as beneficial as possible to evolution and health.
AcknowledgementsFor help with this text I profoundly thank, among others, particularly David Marsh on the history aspect, and Edward Tuddenham, Barry Keverne, and Michael Crawford, without shedding onto them any responsibilities for its inadequacies.
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