Scientists reproduce a stress-induced phenotype in mouse pups thanks to epigenetic reprogramming

© Elena E. Giorgi

I'm excited to be blogging about science again, albeit only occasionally. Those of you who have been following the blog from its very beginnings, back in 2011, know that I've always been fascinated with epigenetics, one of my favorite topics to discuss. So much so that I've managed to include it into the plot of my detective thriller Chimeras. The thrills in the book are fictional, but the science is all real.

I was talking with my colleague Karissa Sanbonmatsu last week, who's been working on RNA and epigenetics since the early 2000s, and she was telling me how the field is still riddled with controversy. There's more and more evidence that environmentally triggered traits like stress, fat storage, and the propensity to acquire certain diseases can be passed on from one generation to the next via activated epigenetic marks, yet many scientists still refuse to believe it. How can things that are not encoded in the DNA be transmitted to the new generation? Germ cells carry epigenetic signatures that have been shaped by the environmental exposures from the parents, but how are these signatures communicated across generations?

A little background.
Our cells carry long bits of RNA that sense molecules and their changes in concentrations. Depending on the environmental exposures they find, they recruit epigenetic factors that then activate certain genes and/or deactivate others. This happens by inducing changes in the chromatin, the big yarn of DNA that sits inside the nucleus. When a gene needs to be activated, the big yarn moves until that particular gene is exposed on the surface and then translated into proteins. On the other hand, to silence the gene, the chromosome move around again and "hide" the gene deep inside the chromatin. RNA molecule act as regulators of these mechanisms, "deciding" which genes to activate and which ones to silence.

A recent study published on PNAS sheds new light on the mechanisms that communicate epigenetic marks from the germ line to the offspring, proving that epigenetic signatures acquired by the parents can be passed onto the offspring. Rodgers et al., from the University of Pennsylvania, used a mouse model to establish the following points:

• First, they exposed male mice to chronic stress prior to breeding, and then observed reprogramming of certain genes in the hypothalamus of the offspring;
• Second, they looked at the sperm of the stressed mice and compared it to the sperm of non-stressed mice; they found a change in content of micro RNAs (miRNAs), and 9 miRNA molecules in particular were found in much higher concentrations in the stressed mice's sperm [1]. Rodger et al. hypothesized that the 9 miRNAs were responsible for the genetic reprogramming induced by the chronic stress exposure and passed on through the paternal line.

To prove it, they injected the 9 miRNAs into single-cell zygotes that were then implanted into normal female mice, raised with no stress exposure, and then examined to see if they presented the same stress phenotype observed in the stressed male's offspring. Indeed, expression of the target genes in the hypothalamus was reduced in the mice that originated from these zygotes, and the expression patterns observed in these mice recapitulated what they had observed in the offspring of the stressed mice.

This study, published in PNAS last october [2], is a milestone in epigenetics, as it finally shows a molecular mechanism that allows genetic reprogramming in the parent to be transmitted to the offspring.

As a final thought, I want to toss in my two cents on the debated rise of autism spectrum and ADHD disorders currently observed in the Western world. Of course, there's the caveat that the diagnostic methods have changed drastically in the past few decades. Still, the increase seems real and the sad truth is that there's probably more than one cause, and the causes lie not just in what the child has been exposed to, but, once you throw in epigenetics into the pictures, his/her parents and grandparents as well. My parents for example grew up at the peak use of asbestos, DDT, and lead in paint. Yes, they survived and, knock on wood, they are quite healthy in fact. But I do fear that we will carry the consequences of those exposures for a few more generations. And who knows what the current exposure to the massive use of corn syrup and antibiotics will do to future generations. Food for thought.

[1] Rodgers AB, Morgan CP, Bronson SL, Revello S, & Bale TL (2013). Paternal stress exposure alters sperm microRNA content and reprograms offspring HPA stress axis regulation. The Journal of neuroscience : the official journal of the Society for Neuroscience, 33 (21), 9003-12 PMID: 23699511

[2] Rodgers, A., Morgan, C., Leu, N., & Bale, T. (2015). Transgenerational epigenetic programming via sperm microRNA recapitulates effects of paternal stress Proceedings of the National Academy of Sciences DOI: 10.1073/pnas.1508347112

Sex Is Always Well Worth Its Two-Fold Cost

Title borrowed from Feigel et al. [1].

Sex is costly. In an asexual population, all individuals bear offsprings, resulting in a higher growth rate than in a sexual population (two-fold cost of sex). Finding a partner is risky, costly in terms of energy and resources, and it results in sexual selection which may not always favor survival. Finally, in sexual populations each individual passes only 50% of its genetic make-up to their offsprings and, furthermore, genetic recombination could break-up alleles that are in an epitastic relationship with one another (they are advantageous when together, but once separated they may incur into fitness loss).

However:

"The advantages of sexual reproduction stem from quite various roots. For instance, sex increases genetic variability by recombination of the parental chromosomes. It makes a population more resistant against many unpredictable threats, such as deleterious mutations, parasites, a fluctuating environment, or competing groups. It also optimizes the evolutionary search for the best gene combinations in a single individual (epistasis) [1]."

Let's try an understand this better. Different alleles in the genome are not always independent, as they may affect fitness in conjunction, a mechanism called epistasis. For example, two alleles may be beneficial together, but their benefit may be lost when separated by a recombination event. Or, it could be the other way around, that a mutation arises under certain constraints, and it's not until paired with a second mutation that it becomes beneficial. This is often observed in drug resistance, for example. A mutation that confers the organism (a virus, or a bacterium) drug resistance could potentially make it less fit (for example, if it makes the organism more "visible" to the immune system). In these cases, often one observes a new mutation arise in conjunction with the drug-resistant one, and the two together restore the organism's original fitness. These secondary mutations are called compensatory mutations because they compensate for the original loss of fitness.

Recombination of genomes can go either way: it can bring beneficial mutations together, or, it can break them apart. In a Nature Genetics review [2], the authors mention a study done on segmented viruses: in this case, "sex" is equivalent to two viruses co-infecting the same cell, as when this happens the enzyme that replicates the genes jumps back and forth between the two genomes and the resulting new genome is a reshuffle of the two parental ones. The advantage of using viruses to study the effect of sex is that you can compare the result of sexual reproduction versus asexual reproduction in the same population. In the case of the segmented virus study, it was observed that an adverse mutation was slower to get cleared in the sexual population than the asexual one.

The same review cites studies done on yeast that yielded mixed results: some showed that sex did increase the rate of adaptation of the population, and some showed the opposite. A paradox? Not quite, if you throw into the picture the size of the population.

"Two recent studies have also tested the effect of recombination on the rate of adaptation in evolving microbial populations. When populations of C. reinhardtii that initially lacked genetic variation were allowed to adapt to a novel growth medium in sexual and asexual populations of varying size, sex increased the rate of adaptation at all population sizes, but particularly in large populations [2]."

Another study done on sexual and asexual yeast strains, compared adaptation in two environments: the mouse brain, which represented a highly variable environment, and a test tube with minimal growth medium.

"When sex was induced, the sexual strain won the competition in the mouse brain but not in the test tube, despite the fact that it also showed general adaptation to this environment. These results indicate an advantage to sex during adaptation to variable or harsh environments [2]."

Despite all these studies, it is still unclear what drove the evolution of sex. Did sex prevail thanks to epistasis? Or was it just drift, the random accumulation of mutations due to pure chance? More recent studies have looked at a combination of mechanisms that may have been responsible for the rise in sexual populations. For example, other aspects to account for, besides epistasis and drift, are redundancy and genome complexity. As organisms have evolved, their genomes have increased in size and complexity. Redundancy allows for more than one gene or pathway to have same function, buffering the effect of deleterious mutations. It also maintains a reservoir of non-coding allele variants that are always available in the search for new evolutionary pathways. At the same time, sex and recombination together cause genomes to be more robust and overcome the short-term disadvantage in favor of long-term advantages like increased evolvability.

[1] Alexander Feigel,, Avraham Englander,, & Assaf Engel (2009). Sex Is Always Well Worth Its Two-Fold Cost PLoS ONE DOI: 10.1371/journal.pone.0006012

[2] J. Arjan G. M. de Visser & Santiago F. Elena (2007). The evolution of sex: empirical insights into the roles of epistasis and drift Nature Genetics Review DOI: 10.1038/nrg1985

Ms. Stick Insect

Image credit: funkman.org.

 
You're looking at a stick insect, a critter I was quite used to growing up as my dad, an evolutionary biologist, used to grow them at home. I know, most households have cats, dogs, guinea pigs and rabbits; ours had cats, dogs, toads, fruit flies, and stick insects. :-)

Children have a tendency to personify everything, animals in particular, so imagine my shock when my dad told me that stick insects are all... ladies. Yup. It's Ms. Stick Insect. And the reason why I mention this is that today I'd like to talk about sex. Ha! You didn't see that coming, did you?

How does an all-female population manage to reproduce? Embryos develop from eggs using parthenogenesis, without the need to be fertilized. This doesn't mean that the offsprings will be identical to the parent. "Reshuffling" of genes is still ensured by meiosis.

In organisms that reproduce sexually, meiosis produces gametes, cells that carry half of the chromosomes and therefore, once fused with the opposite sex gamete, it will produce a cell with the full number of chromosomes. In organisms that reproduce sexually, meiosis produces gametes, cells that carry half of the chromosomes and therefore, once fused with the opposite sex gamete, it will produce a cell with the full number of chromosomes. In diploid organisms (organisms that have two copies of each chromosome), meiosis takes place in the following steps: (i) DNA replication, which creates two exact copies of each chromosome; (ii) pairing of the chromosome homologs, one maternal and one paternal; (iii) the homologs' cross-over creating a unique mix of maternal and paternal DNA; (iii) another round of cell division creates four cells, each with one set of chromosomes.

In parthenogenesis meiosis, step (i) is skipped. In order to restore the two copies of chromosomes, in some perhenogenetic animals, the cell division in step (iv) creates two cells instead of four, each with two copies of chromosomes. However, stick insects employ a different strategy: step (iv) still creates four cells, of which only one has the cytoplasm. This cell then fuses with one of the other three effectively creating and egg with two copies of chromosomes, perfectly equivalent to a fertilized egg.

Not all stick insects reproduce through parthenogenesis. Some populations do have males and mate, though usually only about 10% of offsprings come from sexual reproduction. Morgan-Richards et al. [1] compared several populations of New Zealand stick insects (C. hookeri), and found that while mated females produced male and female offsprings in equal numbers, virgin females that reproduced via parthenogenesis produced mostly females. That's right, I said "mostly".

"A single male hatched from an egg laid by a captive virgin mother. [...] This male may have arisen by the loss of an X chromosome during cell division (non-disjunction), a mechanism recorded for other stick insect species with the same XO⁄XX sex-determination mechanism seen in C. hookeri [1]."

So even in completely parthenogenetic populations, in principle sexual reproduction is not completely lost as the reshuffling provided by meiosis can, occasionally, originate a male offspring. Furthermore, the authors confirmed a geographical distribution of the parthenogenetic population of stick insects compared to the sexual ones: all female populations in New Zealand tend to be more common farther away from the equator and at higher altitudes, implying the adaptive advantage of parthenogens in certain environments but not in others.

The fact that parthenogens would have an adaptive advantage intrigued me, so I dug a bit further and found out about a concept called the two-fold cost of sex. In a sexual population, only one of the two sexes bares offsprings, while in a one-sex population all individuals bare offsprings, hence significantly increasing its growth rate. This seems to indicate that asexual populations have a higher Darwinian fitness. So, how did we end up with so many sexual species given especially that we all originated from asexual ancestors? How can sex be evolutionary successful when the odds seem to be against it?

I'll save that discussion for the next post. :-)

[1] MARY MORGAN-RICHARDS,, STEVE A. TREWICK,, & IAN A. N. STRINGER (2010). Geographic parthenogenesis and the common tea-tree stick insect of New Zealand Molecular Ecology DOI: 10.1111/j.1365-294X.2010.04542.x

Reprogrammable cells

Can't remember if I already shared the above picture... it's my favorite sunset shot so far, so forgive me if it's a deja vu.

The Nobel Prize in medicine this year was awarded to John Gurdon and Shinya Yamanaka for pioneering the reprogramming of cells into an embryonic-like state. Embryonic stem cells are cells that undergo asymmetric division, as they divide into an undifferentiated cell and into a specialized cell. This way, they can grow indefinitely while maintaining their undifferentiated state and, at the same time, keep the ability to differentiate into all three germ layers, the cells formed during embryogenesis.

When still a PhD student, in 1958, Gurdon cloned a frog using the nucleus of a cell taken from the intestine of a tadpole. It took another 38 years before the first mammal was cloned: the first cloned sheep, Dolly, was born in 1996 from an unfertilized egg whose nucleus had been replaced with the nucleus of an adult cell. In this case, the adult cell, by being placed into the egg, was effectively "reprogrammed" into an embryonic stem cell. Up until Gurdon's work was published in 1962, general belief was that once cells specialized, they could not revert. The discovery that cells can actually undergo "reprogramming" under special circumstances is quite significant because it gives hope that we can achieve tissue regeneration and treat degenerative diseases or spinal cord injuries.

In 2006 Yamanaka and his colleague Kazutoshi Takahashi published a paper in Cell [1] in which they showed that, activating four genes, they were able to reprogram adult fibroblasts from mouse embryonic cells. They called the new cells induced pluripotent cells, or iPS, and found that they expressed embryonic-state cell markers. In fact, once in the proper environment, they contributed to embryonic development.

Yamanaka is a strong believer that this research will eventually lead to successful regeneration therapies. In fact, he plans to start a bank of induced pluripotent stem cells obtained from 75 different cell lines. Is this the beginning of a new era? A word of caution comes from a paper published in PNAS at the end of 2010 [2]: in this paper, Serwold and colleagues derived mice from reprogrammed T-cells (cells from the immune system) and showed that roughly half of the mice generated this way spontaneously developed T-cell lymphomas.

The mice were generated by transferring T-cell nuclei into enucleate oocytes. As they mature, T-cells undergo genomic rearrangements, and while normally these rearrangements occur in T-cells only during a specific stage of their development, such rearrangements were observed in all somatic cells in the cloned mice. In [2] Serwold et al. show that these rearrangements undergo T-cell lymphomagenesis: in other words, they cause cancer. Though T-cells are not the only cells that currently can be reprogrammed, this study clearly shows that different cell lines can yield different outcomes, some quite deleterious.

"This study suggests that precautions should be taken to ensure that the identity of the reprogrammed cell of origin is known, and that T cells, and probably also B cells, are not inadvertently turned into therapeutic iPS cells. Recent studies have used human blood-derived T cells as sources of iPS cells, and these cells promise to be valuable tools for studying human immune development and disease; however, the results presented here indicate that extra caution is warranted regarding the therapeutic use of such T cell-derived iPS cells [2]."

[1] Takahashi, K., & Yamanaka, S. (2006). Induction of Pluripotent Stem Cells from Mouse Embryonic and Adult Fibroblast Cultures by Defined Factors Cell, 126 (4), 663-676 DOI: 10.1016/j.cell.2006.07.024

[2] Serwold, T., Hochedlinger, K., Swindle, J., Hedgpeth, J., Jaenisch, R., & Weissman, I. (2010). T-cell receptor-driven lymphomagenesis in mice derived from a reprogrammed T cell Proceedings of the National Academy of Sciences, 107 (44), 18939-18943 DOI: 10.1073/pnas.1013230107

Limb regeneration: a lesson from salamanders

As much as we would love to enlist limb regeneration among modern science's best accomplishments, so far it is still very much confined to science fiction. That doesn't mean it won't happen, though. Key to limb regeneration is cellular reprogramming that allows differentiated cells to return to a germline-like (undifferentiated) state. Genes involved in embryonic development need to be reactivated in order to restart the same process that created the limb during the growth of the embryo.

The vertebrates with the best ability to regenerate limbs are salamanders, making these little critters the most studied in the field. When a salamander loses a limb, a layer of epidermis grows to cover the wound, and beneath this layer new, undifferentiated cells start proliferating, forming a mass called blastema. Recent research shows that this first wave of cell dedifferentiation may recapitulate events occurring during embryogenesis.

"The cells in the limb blastema are believed to be a heterogeneous collection of dedifferentiated cells that have been reprogrammed to achieve varying levels of developmental potential exhibited by the cells involved in embryogenesis [1]."

Germline stem cells are cells that give rise to gametes (the reproductive cells) and have the ability to divide into another stem cell as wells as a more differentiated cell. This mechanism, called asymmetric division, is controlled by a protein called PIWI through small, non-coding RNAs called piRNAs. In [1] Zhu et al. showed that when salamanders regenerate a limb, a germline-like state is established in the growing tissue. In particular, they found that germline-specific genes were expressed in the regenerated limb. In order to show this, they looked specifically at the PIWI proteins.

Zhu and colleagues found a significant amount of upregulated transposable elements in the regenerated limbs. If you remember, transposable elements is a segment of DNA that can move from one locus to another within the genome of the same cell. During the limb regeneration process, transposable elements can impart a deleterious amount of instability, which is counteracted by a corresponding upregulation of the PIWI genes. Conversely, when the PIWI genes were knocked down (i.e. their expression was reduced) in the blastema, limb growth following the amputation was significantly reduced compared to controls.

Zhu et al. conclude

"In the future, further characterization of the subpopulations of these reprogrammed cells with additional germline-specific markers might provide more insight into exactly how far cellular dedifferentiation can proceed and whether there are indeed a small number of cells that could be isolated before a certain developmental threshold and exhibit true pluripotency when isolated from the influence of the partially programmed blastemal cells in the proximity."

[1] Wei Zhu, Gerald M. Pao, Akira Satoh, Gillian Cummings, James R. Monaghan, Timothy T. Harkins, Susan V. Bryant, S. Randal Voss, David M. Gardiner, & Tony Hunter (2012). Activation of germline-specific genes is required for limb regeneration in the Mexican axolotl Developmental Biology DOI: 10.1016/j.ydbio.2012.07.021