Obesity and diabetes: Actual epigenetics or just IVF?

This press release that appeared in my newsfeed titled "You are what your parents ate!" caught my eye because I'm a new mom of a new human and also because I study and teach human evolution.

So I clicked on it.

And after that title primed me to think about me!, the photo further encouraged my assumption that this is really all about humans.

"You are what your parents ate!"

But it's about mice. Yes, evolution, I know, I know. We share common ancestry with mice which is why they can be good experimental models for understanding our own biology. But we have been evolving separately from mice for a combined total of over 100 million years. Evolution means we're similar, yes, but evolution also means we're different.

Bah. It's still fascinating, mice or men, womice or women! So I kept reading and learned how new mice made with IVF--that is, made of eggs and sperm from lab-induced obese and diabetic mouse parents, but born of healthy moms--inherited the metabolic troubles of their biological parents. And by inherited, we're not talking genetically, because these phenotypes are lab-induced. We're talking epigenetically. So the eggs and sperm did it, but not the genomes they carry!

This isn't so surprising if you've been following the burgeoning field of epigenetics, but it's hard to look away. This fits with how we see secular increases in human obesity and adult-onset diabetes--it can't be genomic evolution, it must be epigenetic evolution, whatever that means!

As the press release says...

"From the perspective of basic research, this study is so important because it proves for the first time that an acquired metabolic disorder can be passed on epigenetically to the offspring via oocytes and sperm- similar to the ideas of Lamarck and Darwin," said Professor ...

Whole new ways of thinking are so exciting.

Except when you remember a two-year-old piece by Bethany Brookshire (because you use it to teach a course on sex and reproduction) which explained something that suggests we may have a major experimental problem with the study above.

In IVF, the sperm gets isolated (or "washed") from the semen.

You know what happens, to mice in particular, when there's no semen? Obesity and other symptoms of metabolic syndrome! There are placental differences too. This was published in PNAS.

"Offspring of male mice without seminal fluid had bigger placentas (top right) and increased body fat (bottom right) compared with offspring of normal male mice (left images)" from The fluid part of semen plays a seminal role by Bethany Brookshire.

So I went back to look at the original paper that the press release with the donut lady was about. I wanted to see if they are aware of this potential problem with IVF and whether it explains their findings, rather than the trendy concept of epigenetics...

So even though they titled it "Epigenetic germline inheritance of diet-induced obesity and insulin resistance," I wanted to see if they at least accounted for this trouble with semen, like how it's probably important, how its absence may bring about the same phenotypes they're tracking, and how IVF doesn't use semen.

But I don't have access to Nature Genetics.

Who has access to Nature Genetics, can check out the paper, and wants to write the ending of this blog post?

Step right up! Post your work in the comments (or email me holly_dunsworth@uri.edu, and please include a pdf of the paper so I can see too) and I'll paste it right here.

Update 12:19 pm
Two very good comments below are helpful. Please read those.

I'll add that I now have the pdf of the paper (but not the Supplemental portion where all the methods live and other important information resides). This quote from the second paragraph implies they do not agree with the finding of (or have forgotten about) the phenotypic variation apparently caused by sperm washed of their seminal fluid:

"The use of IVF enabled us to ensure that any inherited phenotype was exclusively transmitted via gametes."

As the second commenter (Anonymous) pointed out below, there does not appear to be a comparison of development or behavior between any of the IVF mice and mice made by mouse sex. So there is no way to tell whether their IVF mice exhibit the same metabolic changes that the semen/semenless study found. Therefore, it is neither possible to work the semen issue into the explanation nor to rule out its effects. Seems like a missed opportunity.

Completely unrelated and inescapable... I'm a little curious about how the authors decided to visualize their data like this:

Epigenetics: what is it and what isn't it? Part I: basic ideas

Epigenetics is a word that has had a variety of meanings historically, and it's sometimes unclearly employed, even by the user.  But these days, when people talk about epigenetics they generally mean the chemical modification of DNA sequence in a way that does not change the sequence itself but affects the expression of genes in or near the modified DNA region--that is why it is called 'epi' genetic. Such chemical modifications affect whether or not the cell uses a particular gene (only a subset of all genes is used in any particular cell, but that subset changes depending on the cell's local environment at any given time). That is, epigenetic changes essentially are regulatory; the epigenetically modified DNA sequences are not mutations of the coding of the structure of a particular protein (or a directly functional RNA), just how or when or how intensely it is used by the cell. Likewise, epigenetic modification doesn't change the affected sequence itself, but affects whether regulatory proteins can bind there to cause a nearby gene to be expressed, that is, transcribed into RNA.  The phenomenon of such DNA 'marking' itself isn't controversial, and a few of the means by which it happens in a cell are known.  Indeed, unlike mutations in the sequence itself, the marking is easily erased and there are known mechanisms that do that.

However, reports that epigenetic marking can be inherited are quite legitimately controversial.  There are a few reasons for this.

How can local gene usage be inherited?
Cells respond to their environment--to extra-cellular conditions--via cell-surface receptors or other similar means.  If they don't have receptors for a signal floating by, they can't detect or respond to that signal. But cells that do detect a signal change whether they start or stop using a particular set of genes. That's how complex multicellular, multi-organ system organisms become differentiated, as well as to respond to environmental conditions.  Most examples of epigenetic inheritance relate to experience that affects particular types of cells, though many 'housekeeping' genes, genes that carry on basic metabolism, are used by all cells, and any environmental change could in principle induce all cells to change their gene usage.

Unless there is subsequent environmentally-induced change, once modified, when they divide, cells transmit their particular expression state to their daughter cells. If an epigenetic modification causes a cell to respond to a particular environmental signal by turning on the expression of a particular gene, that 'use it!' state would be passed on when the cell divides to produce other cells in its lineage, unless or until another modification occurred to reverse the original change. Thus, if some particular cell, say a lung cell, is induced by some environmental factor like a nutrient to express some set of lung-related genes, the effect is local, specific to lung cells. How that works is complex but some of the mechanisms are known.  However, they have to do with how chromosomes specifically in lung cells are packaged; that is a local fact.  For example, it need not also affect nerve or vessel or skin or stomach cells.  Again, that is because in a differentiated organism different tissues are separated from each other so they can be different.

This raises a serious problem: Local effects on gene expression will be passed on to daughter cells in that tissue, but this is not the same as transmitting the effect to the next generation of organisms. Intergenerational transmission requires that the modification also be made in germline--sperm or egg cells--because the offspring organism starts out life just as a single fertilized egg (which has no lung cells!). Germline cells generally need to have genes switched on (or off) to enable them to make a new organism from scratch, from that single fertilized egg cell. Some temporary change that was important to the embryo's future lung cells would not likely be appropriate for the development of those cells in the first place during embryogenesis. So it is no surprise that there are active mechanisms to strip off epigenetic changes in germ cells' DNA, to reprogram those cells' gene usage to prepare them for their embryonic duties, this is done by erasing and re-setting DNA modification in the sperm or egg cell. If the embryo's lungs, when they eventually have them, need to modify what they due based on the air their exposed to, then new epigenetic changes will occur. Thus, the process of erasing and reprogramming removes those changes. Some bits of the genome are protected from this but it is not automatically true that even environmentally induced changes in housekeeping gene usage will be transmitted.

It was first systematically shown by Weismann in the 19th century and has been a theoretical bulwark against the idea of Lamarckian inheritance, that at least in most animals, somatic (body) and germline cells are separated, independent lineages isolated from each other (the situation is different in many or most plants).  That means that for epigenetic changes to become heritable--and hence affect evolution--modifications to particular body cells would have to be applied to germline cells and not be erased before fertilization.

Without some clear mechanism, there is no reason that future sperm or egg cells will even 'know' about, much less respond to, the signal that induces change in the lung or nerve or stomach cells.  So for epigenetic change to be inherited, there is the serious question of how the genomes in germline cells are specifically modified by signals that affect nerve or lung, etc.  If a lung cell alters its use of gene X related to how lungs work, when it detects some (say) pollutant in the air, how does that specific change also get imposed on the germ line?  Explanations that have been suggested so far are mainly not very convincing. That's why most reports of inherited epigenetic modification are properly received with skepticism.

Still, many investigators are seriously interested in epigenetic changes, especially when or if they are inherited, for a few reasons. This sort of inheritance, which modifies DNA usage differently among a person's many different localized tissues, threatens the degree to which traits can be predicted from a person's DNA sequence alone (obtained, for example, from a blood sample), and among other things that threatens realization of the promise of 'precision' genome-based medicine.  Secondly, accurate assessment of epigenetic effects could lead to a better understanding of important environmental exposures and/or what to do about them, so that newborns are not doomed by their parents' habits to live with pre-set epigenetic traits that they now cannot prevent.  And the least legitimate reason, but one important in the real world of today is that is a lucrative and sexy new finding that can be made to seem a melodramatic 'transformative' shift in our understanding of life.

An important criterion for claims of true epigenetic inheritance is that they must pass through at least to a 3d generation without the presence of the environmentally causal trigger.  That is, transgenerational transmission is evidence that the genome is in fact preserving the change rather than just each new individual learning it from environmental experience (such as in utero).  While there have been various generally convincing reports of true transgenerational inheritance in some species like the simple nematode (C. elegans) or plants, this hasn't clearly been shown in mammals (or humans), even if one or even two generational inheritance, usually through the maternal line, has been found.

Most of the literature consists of curious reports or claims of epigenetic inheritance, reviews of the germline erasure process and what areas of germline DNA could perhaps escape erasure of epigenetic marking, and some examples that seem to be truly transgenerational.  At present, the excitement seems generally far exceeding the reality.  But since epigenetics is potentially quite important, and the methods for understanding it rather new, it is being given serious attention.

A paper by Bohacek and Mansuy (November 2015 Nature Reviews Genetics), reviews what is known about the degree to which epigenetic 'marking' is inherited.  This is a very good, measured paper that in our reading of it makes it clear that claims of non-trivial multi-generational DNA modification effects still need careful documentation.  But if life-experience by parents can affect their offsprings' traits in substantial ways related to the offsprings' future life experience, even if they are not exposed to the risk factors that set their parents' genome usage patterns, then if we could understand how this works perhaps such modifications would not be destiny, and means of prevention or control could be developed if the phenomenon were to be better understood.

Gene usage isn't the same thing as gene structure
Epigenetic inheritance can also affect ideas about how evolution works, if they really have long-term (many generational) effects. The suggestion is now routinely being made that the phenotypic effects of epigenetics we are seeing introduces a Lamarckian view of evolution that may, after all, have to be melded with our Darwinian theory (e.g., see Skinner, MK, Gen Biol Evol. 7: 1296-1302, 2015).  But the idea that this is a genuine revival of Lamarckism is still treated with sneering.  Should it be?

We have written a 2015 series of posts about Lamarckian ideas.  Lamarck was interested in the evolution of adaptive traits, like flying or ocean-living mammals, not just some specific minor traits. He had some non-starter ideas, but so did Darwin and they had far less knowledge than we do!  So one can't defend his theory per se for various reasons.  Still, it's worth thinking about rather than just sneering at Lamarck.  That's for tomorrow's post.....

Remembrance of things past--in your genes? Part III: Was Lamarck so laughable?

A favorite sport of those holding to strict Darwinian views (to the extent they understand Darwin),  is to ridicule Jean Baptiste de Lamarck (1744-1829), he of the stretchy giraffe neck.

Lamarck

Lamarck has gotten a very bad name and at least partly undeservedly.  As are we all, he was a product of his time, his academic environment, and the knowledge then available.  He was apparently a quirky personality and got crosswise with other powerful French biologists, notably Georges Cuvier.  For various reasons it became important for Darwinians to distance themselves from Lamarck as an important intellectual ancestor, in particular, to avoid crediting him for his insight about evolution. Intentional PR-spinning to advance Darwinians (and British over French science)?

Lamarck did his best to clarify very explicitly that he was seeking material explanations not being mystical as he is essentially accused of being.  His basic idea of the inheritance of acquired characteristics was not even that new. It was the obvious thing to infer from the data available at the time, and the idea was commonly held as far back as Hippocrates (probably classics scholars can find it elsewhere as well).

In fact, Lamarck was very clear that he wanted a strictly materialistic explanation for species evolution and diversity. This is interesting, and if it weren't for the rather smug glee with which Lamarck is so universally ridiculed by biologists (whether or not they've read his actual work or even much of Darwin's), we might not want to make the following points.  But under the circumstances, we think it's merited, especially in light of the interpretations being given to widespread reports of various sorts of epigenetic inheritance, that is, of DNA marking rather than sequence change during life.

In our two prior posts in this series (here and here), we took the usual view and stated that any suggestion that epigenetic inheritance is Lamarckian inheritance is trying too hard to be revolutionary, because epigenetic inheritance is imposed by the environment, not by some mystic inner drive on the part of the organism as Lamarck is supposed to have suggested as the cause of adaptive evolutionary change. So, as the usual view has it, even if genome marking is inherited, it is not Lamarckian.  But is that actually so?

"Laws of Nature": who was right?

As we use the term, a Law of Nature is a concept that grew out of the so-called Enlightenment period in European culture history beginning around the mid-1600s.  Darwin's view of natural selection was that it was a Law of Nature that Isaac Newton might have been proud to recognize.  In the amended introduction to the 6th edition of the Origin of Species, Darwin added a review of the history of evolutionary thinking, and there he couldn't have expressed his views better: Darwin said that Lamarck "...did the eminent service of arousing attention to the probability of all change in the organic, as well as in the inorganic world, being the result of law, and not of miraculous interposition."  (italics mine).  

But in fact Darwin was quite wrong, reflecting his own ideological commitment, not Lamarck's. This is because Lamarck said something far more important in my view than the way Darwin thought, and in fact the exact opposite. Here's a fundamental point that Newton himself made very clear, about the central characteristic of a Law of Nature: Principia in 1687: 

"Those qualities of bodies that . . . belong to all bodies on which experiments can be made should be taken as qualities of all bodies universally."

That is, if you find something to be true in a local, restricted setting or 'sample', such as dropping an apple or the orbit of the moon around the earth, the same would be true everywhere else that you didn't or couldn't study.  That was the very essence of what it meant for some phenomenon to be a 'law'.   In his books and other writings it is repeatedly crystal clear that Darwin accepted this Newtonian view: natural selection is a law of nature the way the law of gravity is.  Indeed, in the autobiography he penned for his children near the end of his life, he couldn't have been more clear, writing "...now that the law of natural selection has been discovered..." and "Everything in nature is the result of fixed laws.”

Lamarck was closer to Newton in time than Darwin, but what were his views about laws of nature?  I know not, but Georges Cuvier gave a scathing 'eulogy' upon Lamarck's death, a bitter attack that poisoned posterity about Lamarck's reputation, and Cuvier notes of Lamarck "He had meditated on the general laws of physics and chemistry, on the phenomena of the atmosphere, on those of living bodies, and on the origin of the globe and its revolutions."  If accurate, Lamarck shared the prevailing idea of laws of Nature with Darwin.  Yet, when it came to life, Lamarck in his book said something very cogent, that Darwin and his intellectual descendants seem not fully to realize, even to this day: 

"In dealing with nature, nothing is more dangerous than generalizations, which are nearly always founded on isolated cases: nature varies her methods so greatly that it is difficult to set bounds to them."

Lamarck wrote this basically before the widespread development of statistical thinking, but it is a fact that has still not yet been absorbed by most biologists in evolutionary or biomedical genetics. Lamarck said that when the environment changes, that change in turn induces responses in behavior of organisms. His theory was about the consequent importance of (1) habit, (2) the use and disuse of traits, (3) the inheritance of acquired characters, and (4) the very slow process of adaptive evolution. 

As Lamarck described things, organisms have ways of life that depend on their circumstances.  They seek out resources, like food, that they are able to find, and the resulting 'habits' are essentially their ways of life.  Traits that are used seem to become more important over time but traits that are not used seem to wither or disappear.  Traits acquired during life are passed down to descendants.  The process is very slow, almost unimaginably so.

None of this seems to be at all forced, invented, or strange, and in fact, Darwin adopted all of these ideas in his own way.  As noted above, the idea that one's characteristics were controlled by some sort of transmitted substance is ancient, and the idea of evolution of species was hypothesized in classical times and here and there after that.

[If you are skeptical of our take on this, given Lamarck's clown-like image (due in large part to Darwin and his pals and Cuvier), that would not be surprising.  But that image is wrong, as you can see if you check his book itself and the Introductions by two highly respected evolutionary scholars (the 1984 U of Chicago Press English translation of Lamarck's Zoological Philosophy itself with very informative introductions by Hull and Burkhardt.), or Stephen Jay Gould's The Structure of Evolutionary Theory, or Ernst Mayr's The Growth of Biological Thought.]

So, what was so laughable about Lamarck?

Lamarck is routinely sneered at because, among other things, Darwin and his colleagues were motivated essentially to claim more credit for evolutionary ideas (I'm not the first to suggest this). Lamarck was a human with all the associated failings, but his work is derided because he suggested that the very striving or habits of life caused the associated heritable changes.  By contrast, the Darwinian idea is that new variation arises randomly relative to any need it might or might not have (one can debate how clearly Darwin understood or held such a view).  

However, Lamarck was trying to explain the same phenomena as Darwin, and to do so in terms of natural, historical evolutionary processes, rather than individual events of divine creation.

In our two previous posts in this series we basically took the Darwinian view, that Lamarck was laughable and any attempt to say that epigenetic inheritance was Lamarckian was equally wrong, trying too hard to challenge standard evolutionary theory. In fact, one can argue that epigenetic inheritance really is Lamarckian, based on what he actually said rather than what Darwin said about him, and adjusting for what was known in Lamarck's time.

(1) Habit: How do epigenetic changes arise?  
They arise because of the conditions and behavior of the organism: where they live, what they eat, stresses they are exposed, etc., and how their bodies respond to those exposures.  That is, they are the effects of the habits, as one could say, of the organisms.

And such changes are obviously adaptive if they allow the organism to persist and reproduce! If epigenetic changes are important and persist, over time they will be built into the characteristics of the species. Indeed, there are means by which such traits can eventually be built into the genome in the usual DNA-sequence way (one term for this is 'genetic assimilation').  Over time, nothing strange need be involved for epigenetic changes to be wholly compatible with our understanding of evolution.

(2) What about use and disuse?
In modern theory, 'disuse' means that eventually mutational or gene-expression changes (even if due to epigenetic mechanisms) lead a function or a gene to become more degenerate--as Darwinians would say, because there's no selection pressure to maintain it or even because it's costly if not useful and selection will favor its disappearance. And 'use', of course, would mean of adaptive value.  All perfectly compatible with Darwin (and part of his own theory).

(3) What is epigenetic inheritance?  
When and/or if it occurs, it is the modification of DNA (or the contents of cells) that arises in gametes (sperm or egg) during a parent's life and is transmitted to offspring.  The modifications of interest affect gene usage and hence the traits of the organism.  That is, this is the inheritance of acquired characteristics.

(4) What about the pace of evolution?
As to time, Lamarck was every bit as clear about the slow, gradual nature of evolution.  Both stressed this, recognizing the need to avoid creationist explanations.

So many of Lamarck's basic  ideas were similar to Darwin's (again, historians, not just I, have pointed this out).  What matters is not what someone said 200 years ago.  Instead, the bottom line is that if transgenerational inheritance by way of epigenetic changes acquired during life occurs and is functionally relevant, it is basically Lamarckian, but is also just a different form of 'mutation'.  And the differential proliferation of successful inherited traits, however acquired, will be a natural form of selection.

In that sense, it is Lamarck who is being misrepresented, and whose work in this context, given his context, is not risible.  It doesn't make Lamarckism entirely 'true'; there are wildly wrong things in Lamarck (but also in Darwin).  Cuvier, himself grossly wrong about life in many ways, cruelly portrayed Lamarck as a real nut case.  Despite Lamarck's sometimes free-wheeling ideas, that is not the judgment of history, and in any case, based on what Lamarck wrote in regard to the issues here, if epigenetic inheritance does turn out to have long-term relevance, which is not yet the case in terms of current evidence, it does not in any serious way undermine Darwin. What it does do, is to undermine ideological Darwinism.  And that is a very good thing for science.

Remembrance of things past--in your genes? Part I: Is epigenetics 'Lamarckian'?

Marcel Proust's epic novel, In Remembrance of Things Past, was a 20th century masterpiece of thoughtful reflections.  It is about small things that trigger recollections of events that happened earlier in life but that were otherwise lost to memory (an alternative title translation from the French is In Search of Lost Times).  For Proust it was the madeleine, a small cake he dipped in tea (the most famous example), or any number of other unexpected nostalgic triggers.  These evoke times past without one explicitly trying to dig into one's memory: they just come up.  We all have that experience, I assume. An odor, a piece of music, a food trigger a particular birthday or Christmas or girlfriend's name, or event at the beach.  In some instances, Proust's protagonist remembered things about his parents or other relatives.  But wistful as such memories were, they are things in the past that can be retrieved in memory but not in reality.  Or can they?

Forget the parents?
I'm not talking about, say reincarnation or strangely eerie experiences like deja vu. Instead, I'm thinking of reports over the years, and increasingly these days, of genomes that 'remember' events that happened in their, and their ancestors' past.

The consensus idea about genome evolution, supported with overwhelming evidence of all sorts, is that genetic mutations, that is, changes in DNA sequence, occur through various chemical process that are random with respect to any functional effect they may have.  It is chance and various forms of selection (see our series of posts on these, starting here), that determine which changes proliferate over the generations.

Before the understanding of the nature of genetic inheritance, going back from Darwin's wild guesses about gemmules, to Lamarck's famously dissed ideas about the inheritance of acquired characteristics*, even to Hippocrates, was the reasonable idea that your traits were somehow the result of elements (now we would call them 'molecules') that traveled to your gonads to be transmitted to offspring.  They were molecular images, so to speak, of who you were.  It made sense, but Mendel's work and much else showed clearly that that is not how inheritance works, or evolves.  Lamarckian thinking of that sort is out, and not because the Mendelians are bullies, but because there isn't any evidence for it!  Or is there?

Lamarck redux, or trying too hard?
There are many incentives for scientists to try hard to be the next Darwin, and to press their hopeful ideas to the public.  But most such claims are hopeful monsters, quickly shown not to be true. For example, ten years ago two Purdue plant geneticists published a paper in Nature that reported that the plant Arabidopsis, related to mustard, had a self-correcting mechanism that for generations could restore 'good' gene versions, replacing 'bad' mutants.  This if true would be a kind of Lamarckism, in that the organism could remember what was good and impose it (as I recall, the authors were not claiming to be resuscitating Lamarck, but that this was a new or different kind of inheritance).

It quickly turned out that the results were due to some experimental artifact, and pollen contamination and/or other problems undermined these results and nobody, probably not even the original authors, believes them any longer (the authors have tried to suggest that the artifacts didn't explain everything, but this hasn't been convincing and not even the authors seem to be following it up).  Nice try, but no cigar.

At the same time, over recent years, there have been findings that suggest that experiences acquired during life, that involve gene expression, could be transmitted to offspring, but were not encoded in DNA sequence.  Instead, they were epigenetic, that is, they involved modifying the DNA sequence in a way that affected which genes were being used in given contexts.  Clear examples involved coat color genetics in mice and some physiological responses related to obesity and associated traits.  The idea is that a fetus could acquire the change in gene usage while in utero, which would affect its traits, and then 'remember' the gene-usage setting and transmit it to their offspring.

There have long been suggestions that offspring can, even when adults, have physiological traits that resemble their parents, not through inheriting genetic variation but inheriting physiological states themselves.  Examples included traits like blood pressure, where mothers' state during pregnancy set their children on a related path, such as to having high blood pressure.  Terms like 'set point' were used to describe how the infant's body was 'set' to respond to its life experience in a way that resembled how the mother's state was, but that was not because of her specific genotype.  These results could not be related to known genes at the time, but technology has improved and there are several examples and some of the genetic basis seems to be becoming known.

Epigenetic changes are real
In short, what we know has to do with gene usage, not gene sequence.  Gene usage is affected by very well-known mechanisms that modify the chemical state of a given DNA region in ways that enable a nearby gene to be used (or, depending on the mark, prevent usage).  This is known as epigenetic marking because it is not due to mutations in DNA sequence.  The difference is basically that between Lamarckian and modern inheritance ideas in that epigenetic changes can be directed--that is, set or removed--based on experience.  Generally, the idea is that epigenetic changes are responses that relevant cells 'know' how to make in a given environmental context.

These effects thus seem 'Lamarckian' in a restricted sense, but that has been thought to be very restricted.  Your body's cells, say muscle or heart cells,  may respond to their environment (e.g., what's passing by in the blood stream) by context-specific mechanisms that use epigenetic marking to turn some genes on or shut others down.  This may be inherited by the body's cells, when they divide, unless changing circumstances lead them to change the genes they're using.  But that would not necessarily be inherited, because all you transmit to your offspring is a sperm or egg cell.  There is no reason to think that, for example, nutritional components that affect how insulin is used or how fat cells store energy would affect the use of energy-storing genes in sperm or egg cells.

This might happen, however, if the body cells, or even all the cells of a fetus including the germ line, also sense the maternal environment, and that in turn induces similar changes in the fetal cells.  The physiological settings of the fetus would reflect the mother's experience, not by being transmitted in her egg cell but by the effects of her blood constituents via the placenta.

A fine review of the state of knowledge as of 2014 is by Heard and Martienssen ("Transgenerational Epigenetic Inheritance: Myths and Mechanisms," Cell, 3/27/14, p99). This post today and Monday's post take some selective bits from that, but if you are interested in this subject, it seems to be an excellent source to read carefully.

This figure shows how exposure in utero can transmit to the offspring (F1 generation) or grandchildren (F2), but only if it also appears in the next generation in unexposed individuals is it really now incorporated into the genome for future generations.  The figure labels stress and nutrition as possible causes of gene-expression change that could get into the germline.

Figure from Heard and Martienssen, 2014, Cell

If altered epigenetic settings affected all the fetus's cells, then the settings could be inherited by its offspring, that is, the mother's grandchildren.  Even with no further environmentally induced effects, this could indeed be transmitted for multiple generations, and be more truly 'Lamarckian' inheritance into posterity. Unless the pattern really shows up in the 3d generation, it will not be considered truly transgenerational.  But even then, or with the examples, there are at least three problems to consider.

First, we know that these set-points are generally changeable during life.  Circumstances set and un-set them, as cells respond to their environment.  Indeed, epigenetic changes are in large part responsible for how tissues differentiate--into stomach, lung, brain, skin, etc.--during embryonic development.  That's how you become a differentiated organism.  Having gene-usage too rigidly programmed during an adult's life (the parent), could prevent the offspring from even becoming an offspring.  So there is likely to be a re-set mechanism so a fertilized egg can start life anew.  Why would your Mom's experience override your own responses?

Secondly, the evidence to date suggests, at least, that even the persistent marking that's been observed fades or disappears eventually.  It is not as permanent as changes in the DNA sequence itself (the genes proper).  The problem that raises is that it won't be part of long-term evolution.  Unless--unless a phenomenon called 'genetic assimilation' occurs.  That's when something not engraved in DNA persists for whatever favoring reason and eventually some 'real' mutations--DNA sequence changes--with similar effect arise.  In that case, the actual hard-wired changes can persist, with the 'good' trait being produced even when the epigenetic marking has long gone.  But that then is a form of ordinary rather than Lamarckian selection, and how often would such things arise in the world? This has been debated since CH Waddington's advocacy of the idea as an important evolutionary process, in the mid-20th Century, and indeed the idea was proposed in the late 1800s before any actual genes were known, in a somewhat different context and known as the Baldwin effect.

Thirdly, and at least as important, too much epigenetic change could prevent new mutations--'real' evolutionarily relevant change--from having effects, if gene usage patterns, which are an important part of evolution, were too rigidly entrenched by DNA marking.

Fourthly, sperm and egg cells are developed in particular cell lineages in a fetus, initially called primordial germ cells (PGCs), cell lineages that are isolated from cell lineages that form the rest of the body, and vice versa.  So how is it that something specifically affecting gene usage in a particular organ, like a blood vessel, kidney or eye, would also be 'set' in the gonadal cells?  How exotic would such an information-passing mechanism have to be, and if it exists would we have to re-think our skepticism about Lamarckian inheritance--because if such specific mechanisms exist, they would really be the transmission in the patrimony, of things acquired by experience during life?

In fact, one recent paper has suggested that this may be occurring.  Male rats exposed to a particular odor that is known to activate one particular odor-recipient ('olfactory receptor', or OR) gene and at the same time exposed to a mild fear-inducing stress, were conditioned to activate that OR when exposed to the stress. These males were mated to unconditioned females, and the investigators report that the offspring males respond strongly to that odorant (via the specific OR gene).  But when such offspring males, who had not been exposed to the conditioning fear stress, were mated with unconditioned females, the next, grand-rat, generation also showed the preferential usage of this OR gene.

This latter study is remarkable if you think of the fact that a rat has about 1000 different OR genes, two instances of each (one inherited from each parent), and they are scattered in sets of varying numbers across most of its chromosomes.  So how is it that the rat's body 'knows' how to just mark that one particular OR gene, not just in its nose cells but also in its sperm cells, so the marking can be inherited?

If this experiment is to be believed, and it is remarkable enough that it must be carefully tested, and the effect shown to last for further generations, then we have to wonder what mechanism might underlie it. Is it really Lamarckian?  There is a sort of precedent, in the worm C. elegans, in which olfactory gene switching can be inherited for multiple generations and may have adaptive function.  But if this is true in mammals, is it general enough to challenge the axiom about inheritance on which current evolutionary theory rests?

Adaptive function would seemingly be something a species would 'want' to be permanent and not easily erasable or, put in a more mechanistic way, the freeze-in-place mechanism would eventually work against adaptation if circumstances changed.  And how would such decision-making characters work?  Wouldn't genetic assimilation remove this from epigenetic control?

Some new papers in the June 4th issue of the journal Cell raise questions, or perhaps raise serious problems, about how to interpret these various results on gene-usage effects and their inheritance, and we'll talk about them tomorrow.
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*Lamarckian inheritance has been badly misunderstood, as we'll discuss in part 3 of this series later this week.

Epigenomics: the germ of an idea

The 19 February issue of Nature has many papers reporting a major human epigenomics project (other Nature journals have other papers from this project).  We cannot summarize all of them here by any means, and haven't read all the papers, which range far and wide.  But the growing awareness of and, more significantly, serious attention being paid to DNA usage rather than sequence modification as an important aspect of biological function is worth discussing.

A recent article in Aeon by Guthman and Mansfield discusses the conceptual way in which epigenetics, shows that organisms (including humans) are not closed vessels, whose nature is strictly determined by its carefully sequestered DNA sequence, the individual's insides isolated from their environment and its effects.  We knew, of course, about injury, infection, and lifestyle effects.  But what is becoming apparent is that there probably are far wider ranging effects on our cells that are inherited when they divide and hence become functional parts of our genomics.

Epigenetic mechanisms; Wikipedia

The DNA in our genomes contains codes for the amino acid sequence of proteins among other things. But in a given cell, a given protein code is only used when other non-coding sequence elements, called regulatory elements, near the coding part are properly activated.  This is known as gene expression, or gene usage, and is based on specific proteins binding these regulatory elements.  That in turn depends on the environment of the cell within the body.  During our lifetimes, from conception onward, some aspects of our internal physiology alter a cell's gene usage to suit current circumstances.  This can be due to internal feedback, such as during hunger or sleep and so on.  But for a bit of coding DNA to be expressed, or not, depends on the attachment, or not, of particular chemicals to its regulatory elements.  This modification is called epigenetic, because it affects how the DNA is used without changing its nucleotide sequence.

Some epigenetic changes, and their resulting effects on gene expression, seem to be responses to environmental exposures of whatever sort.  These then can affect 'normal' traits but also may lead individuals to have traits ranging beyond normal to pathogenic.  The longer one is exposed, or the earlier, the more likely a complex of epigenetic changes may, like the simultaneous accumulation of mitotically inherited somatic DNA mutations, affect traits.  That is, one might expect epigenetic traits to show an accelerating age-of-onset distribution.  Of course, this would mainly apply to steady exposures throughout life, or to responses a person is programmed in gestation to make to environmental experiences.

It is somewhat fashionable to refer to epigenetic changes as 'Lamarckian'.  In the early1800s, Jean Baptiste de Lamarck suggested that evolution's slow path was driven by its life-ways.  As organisms strove through their behavior to survive and reproduce (this need not be 'conscious'), that behavior modified the organism's transmitted material (genes, of course, were not known), and this led to a continual adaptive track over eons of time.  Lamarck is accused of mysticism because he suggested that striving influences one's inheritance, but he didn't really mean long-term goal-directedness, just trying to get along in one's environment.  Still the organism was more 'proactive', or had more 'agency', to use modern catchwords, than in Darwin's more blind mechanical process of natural selection by which randomly arising variation was screened for fitness.

In a fundamental way, epigenetics does not necessarily imply any sort of Lamarckism.  If life-ways modify DNA expression in cells and that is transmitted when those cells divide, then gene expression patterns acquired by experience are inherited--but one would expect this to be only somatically: exercise might affect epigenetic changes in, say, muscle or heart or lung cells.  Diet might change intestinal cell behavior.  When those cells divide, the epigenetic marking may be transmitted. In that sense, from the point of view of gene usage, we are not what we inherit, except to the extent to which our DNA sequence affects the epigenetic-marking mechanisms of a given environment.

This sort of leakage between a person's bodily integrity and the environment may be very important in terms of who we are during our lifetimes, and the diseases we get or resist.  This could be called 'Lamarckian' at the somatic (body) cell level, so long as one didn't attribute any sort of mental striving to it.  Of course, brain cells might be modified by such striving.  But that's a kind of loose or careless talk that doesn't reflect an accurate understanding of Lamarcikan evolution, which this is not--and yet....

Is it Lamarckian?
For a Lamarckian sort of evolution, the effect of the environment must be imprinted on the genome either before fertilization or during gestation so that the change is transmitted to the next generation, in the germline (to be incorporated in sperm or egg cells).  This is why much interest attaches to epigenetic changes that may occur to a fetus because of a mother's lifestyle during pregnancy.  This is important because then the fetus may carry the change in all its cells, including its own germline cells, which could make it heritable by its offspring.

Epigenetic changes can be actively imposed or erased, so inheritance of these sorts seems generally to be less permanent than changes in DNA sequence.  The latter can be created or erased, by mutation, but that is a much slower process that only happens to individuals and is far less likely to be erased (except by natural selection if harmful).  Epigenetic changes, even in utero, may be common if many or most mothers are exposed to the same environment or behavior.

Even so there are reports, that clearly need to be made rigorous and not exaggerated by scientists or the media, of multi-generational persistence of epigenetic changes that arose in adults.   The effects of famine in Holland during World War II seem to be an example of heritable epigenetic changes as a result of environmental conditions, effects that are still being seen in grandchildren of women who had insufficient calories during pregnancy.  The effect crossed the placenta and altered the fetus' gene usage.  If such a multigenerational effect prove true, the evolutionary, or trans-generational effect must be marked not just on the person's body cells, but on the germline, too.  For example, if exercise-induced changes in muscle-cell gene usage occur, how could such specific change also be imposed on that person's sperm or egg lineages?

There should be a heavy burden of proof on anyone arguing, as some do, that epigenetic changes due to post-natal behavioral or environmental experiences that do not seem genetic (that is, are not encoded in the DNA sequence) are inherited.  If something dietary stimulates epigenetic change, that may affect all cells.  But (to cite one recent claim) epigenetic response to specific odorants by nasal epithelial cells have no obvious way to be imprinted on the exposed animal's sperm cells.  Yet one study reports pairing a pain experience with the expression of a specific odorant receptor in adult males, and that same expression change was seen in his offspring, and then in the grand-offspring, but without further conditioning.  How does the sperm-cell lineage in the rodent's body 'know' to mark a particular receptor gene out of the many hundreds in its genome?  We need a mechanism if we're to believe the report!  That doesn't mean there isn't one, of course, but it seems highly questionable, because what might it be?

Let's put it this way:  If such histologically 'local' (tissue-specific) experience-targeted changes really can be imposed on the germ line, and that can truly persist for multiple generations, then we really may have something different!

There are many reasons why Lamarckian inheritance was rejected after Darwin's and Mendel's work were united a century or so ago.  Lamarck wasn't being foolish, but he did seem to miss the important point that biological traits arose via random mutation, rather than being directly applied on patrimony by experience.  Natural selection screening existing variation is an easier mechanism to understand. Lamarckian inheritance doesn't necessarily lead to species' extinctions, because they can just modify what they do based on their conditions, whereas in Darwinian evolution, most lineages go extinct (that isn't formally necessary, but it seems to be true, and the generic explanation is that the species was too specialized or simply didn't experience adaptive mutations when environments changed).

Still, we can't be dogmatic about accepting Darwin and rejecting anything else.  Epigenetic mechanisms do seem to provide some nuance to our understanding of inheritance.  Chasing down instances and mechanisms at the cell level is a hot fad right now, and it will probably die down to its useful aspects as most fads do.  But doubt is important when it comes to claims of changes not directly related to germline cells to be imposed on those cells would require much more internal cellular communication than we currently understand. On the other hand, life is to a great extent all about signaling among cells, and maybe there is something going on we haven't understood.  Time will tell but meanwhile circumspection is called for.

The anti-Lamarckian gut reaction: keep the bar high--but your minds open

Biologists tend to ridicule Jean-Baptiste Lamarck for his version of evolution, expressed in his 1809 Philosophie Zoologique.  Laughing at him is one of our favorite sports.  Of course most of those who do the laughing never bothered to do any actual reading of Lamarck's famous book, but who's gonna sweat the details?

So what was it that he said, and why was it so risible?

Larmarckian Inheritance

Jean Baptiste de Lamarck

About 50 years before Darwin's famous theory was published, Lamarck explained the diversity of complex organisms by what we term the inheritance of acquired characteristics.  In his theory, traits that were used by an organism were transmitted to its offspring, and traits that were not used were not transmitted.  Very gradually, organisms would develop, refine, and elaborate useful traits.  Lamarck may have been wrong but not entirely so, given the data and attitudes of the time, and he was seeking a material explanation for biological complexity and its origins.  Good overviews can be found in the prefatory material to the 1984 English translation of Philosophie Zoologique published by the University of Chicago, and by SJ Gould's 2002 The Structure of Evolutionary Theory.  Other authors seem typically more derogatory and pejorative and less clearly acknowledging what Lamarck actually said.

The point is not to defend Lamarck, but to see why his views evoke such gut negative reactions.  The modern purely materialist theory of evolution (largely of Anglo-American origin) stresses randomness of inherited change due to external forces of mutation, and the brutal screening by natural selection to favor those genotypes that confer advantageous traits on their bearers.  Self-satisfied in what really is somewhat our own arrogant dogma (see our series on the mythology of natural selection, which begins on Monday of next week), we brook not even the slightest breach in our own dogma.  Still, in the way he tried to explain things, Lamarck does seem to have been mistaken, and his ideas (which were in the air at the time) in a way set the table for a better kind of material explanation for evolution and the traits of organisms, due to Darwin, Wallace, and a few others who ventured correct partial statements of things.

Lamarck's idea was that what organisms do during their lives, in response to the challenges of their environment, is somehow materially transmitted to their offspring.  This is an inner rather than outer source for the variation: systematic habit-induced physical change rather than externally screened random variation.  It didn't help Lamarck that the Soviet era genetics tried to appropriate Lamarckian rather than Darwinian evolution for various ideological and sociopolitical reasons, nor that some of Lamarck's contemporary rivals denigrated him, nor that Darwin found basic holes in the idea (despite holding a very similar theory of inheritance). 

Our point here is that there are some very good reasons based on 19th and 20th century biology, to hold the view that Lamarck's mechanisms don't hold much water, and this is why when one argues for anything that might seem even the slightest bit Lamarckian, the ridicule begins and the burden of proof is raised to a much higher level than when biologists venture their routine Just-So stories about how the elephant got his baggy skin.

What we do know
One thing we know very well is that gene usage does involve experience-based feedback onto cells' genomes. Cells use a subset of their genomes, and which subset is dictated by their context--by aspects of the cell's local environment.  Indeed, cells are loaded with environment-sensor (like receptor molecules) that monitor the world outside the cell and adjust gene expression accordingly.  This is clear, experience-based modification of the genome.  Of course, the modification is epigenetic:  it is not a change of the code book (the DNA sequence) itself and that is the key.  It is a change in the DNA molecule that affects how it's used, but not the sequence-based code.  To oversimplify, it is the binding of a molecule to a specific sequence-based bit of DNA near a gene that affects whether that gene is used by the cell at that time. When circumstances change, the molecule may leave the DNA, changing whether the gene is used or not.  Because the molecule binds to specific DNA sequence, epigenetic change does involve DNA sequence, but doesn't change it.

Second, in many if not most multicellular organisms the experiences of life affect its body tissues.  If a vertebrate does hard work, its muscles and joints may gain improved strength--something all of us know very well.    But if cell structures are produced by genetically coded molecules, the code itself isn't changed by the experience.  More importantly, an entirely separate line of cells, the germ line (sperm, eggs, pollen, etc.) is separated from the rest of the body's cells early in development.  Pumping iron may pump up your pecs, but it doesn't alter the relevant genes in your germ cells.  Even if it would be a good thing, the basic idea for over a century, supported by lots of evidence, has been that there is no way for a (say) muscle-specific genetic change to be built into the genome of a muscle cell, much less into a germ cell.

Instead, if pumping iron is good for your reproductive success, then those who by good mutational luck carry muscle-related genetic variants will reproduce better, passing the screen of natural selection and proliferating their good genes into the future.

Arnold Schwarzenegger in Pumping Iron; Wikimedia

The problem with a Lamarckian genetic alternative would be that pumping iron selectively mutates the muscle-related gene and when it turns out to be useful then engineers that same mutation in his germ cells. There is no known mechanism by which a specific mutation in a specific useful gene can be engineered into a germ cell, just because it happened to be useful in a muscle cell.  If it exists it will have transformative effect on biology, and that is why the standard of proof is so high, and skepticism so great against anything that seems like such a claim.

In fact, there are many examples in the literature of trans-generational transmission of epigenetic states. Each has been blasted and for essentially this reason:  biologists are generally not willing to open a breach in their Darwinian selectionist firewall.  Since anything truly Lamarckian could threaten the most solid bits of biological-evolutionary theory, it is entirely appropriate that a high bar and burden of proof be maintained.  But if it's true, it really is no threat to the state of the world; it's just knowledge of a new mechanism for at least short-term adaptability of complex species. In fact, it was anticipated way back into the late 19th century.

Current contentious examples

We write this because a report has just appeared in Science that claims something that seems like Lamarckian inheritance.  In this example, a starvation induced in laboratory mice when they were pregnant led to an epigenetic change in various genes that, the authors say, led to risk of diabetes, and that risk also was characteristic of the next generation, the grandchildren, even though their mothers were not subject to starvation. As described in a commentary in 10 July Nature, the experiment is in effect a test of the aftermath of a winter of starvation in Denmark in 1944-45.  Children conceived during this time were born underweight, and experienced health problems which their children, too, seemed to go on to experience.  

This report was just the most recent of several related to claimed epigenetic transmission of chronic disease-related traits that have appeared.  An even eerier recent report claimed that male mice exposed to fear-stimulus in the presence of a specific odor, were conditioned to hyper-express an odor-receptor gene that detected that order, but then this same gene was marked for over-expression in their offspring, and their grand-offspring, even though there was no further fear-conditioning.  (We blogged about this here.)  Gene expression induced by experience in the nose and then the same gene primed in sperm cells to be expressed in the males' offspring--and maintained in the sperm-line for a third, grandchild, generations?  Are you kidding?  

And there are other reports of similar multigenerational epigenetic transmission, some of them in controlled experimental settings like these examples.

The key reason for the strong skepticism at reports like these is that a specific gene in a germ cell line, a cell not directly affected by the environmental factor, is modified by that experience and the modification is then transmitted.  This might not seem like a problem, except that, genome-wide, the epigenetic state is generally highly programed for embryonic development, with sperm and egg genomes are subjected to heavy genome-wide epigenetic reprogramming before conception.

The idea that an experience-based epigenetic responsive state can get into the germ line and be transmitted for several generations is a threat to a Darwinian dogmatist--emotionally, it's like trying to get a biblical literalist to accept that Genesis might be at least a bit metaphoric.

Science is about learning new things
But we shouldn’t just defend dogma by being dogmatic!  There really shouldn't be any problem at all with this kind of multi-generational transmission--if it's true.  If it is, then we just have learned of a new mechanism of adaptability by organisms by which they change their biological state to reflect their circumstances (like shivering when it's cold, or an adrenalin rush when frightened).  Somehow, the body would know which gene was modified epigenetically by experience, and finds that same spot in the genome of a sperm or egg cell and makes the same modification.  That this could be transmitted to future generations could be a fine adaptive mechanism because circumstances might not change, and organisms would be epigenetically prepared from birth to meet them.  If true or shown somehow to be general, we'll all say that this is a marvelous aspect of evolutionary adaptation, and how could we have missed it!  

One can conjure up various ways this might happen….except for the minor detail that we don’t actually know of any such mechanism!  That doesn’t mean it can’t exist but it’s proper that one must find it before the results will be accepted.  Still, the more results of this sort that are reported, the harder should be the search for something that a century of work suggested didn’t exist.  Can all these reports be wrong?  If not…..what can make them true?  If so, are they wrong for some murky methodological artifact?  At some point, sneering should stop and hard work to find the mechanism should start.

We've tried to outline what the controversy is and why it is reasonable to be very skeptical of these reports, that seem so 'Lamarckian' even if we take into account that he was writing, or guessing, based on the state of knowledge 200 years ago.  If indefinite proliferation of an epigenetic change is ever proven, then it will show that this sort of inheritance, even if not affecting DNA sequence directly, is a part of evolution. It won't be exactly Lamarckian, but it won't be exactly Darwinian either.  It will be a remarkable revelation whose discovery we'll celebrate.

But if it's warranted that we give these reports a very hard look, that should not require that we cling to a cartoonesque oversimplification of the current dogma, that everything about everything is the result of Darwinian style competitive natural selection.  And with this comment, we'll return to writing our series about selection and the origin and genetic basis of complex traits.