Too much phosphorylation, time to go to sleep!

It’s Friday night and you are at a concert, wishing you hadn’t woken up at 4:45am to go to spin class. As the night wears on you get more tired and fall asleep on the train ride home. Why do you get tired the longer you stay awake? It’s not your muscles-- they could keep contracting. There are chemical changes to molecules that accumulate the longer we stay awake and they drive this need for sleep. This was shown in a recent paper in Nature by Wang et al. using an interesting mouse mutant.

Sleepy mice

When mice are sleep deprived, they have an increased need for sleep (just like humans who get more tired the longer they stay awake). Sleep need is measured by putting electrodes on the mouse’s scalp that measure brain waves, which are large synchronized and rhythmic patterns of electrical activity in the brain. When mammals sleep, there are characteristic changes in the brain waves, so we can tell what stage of sleep the animal is in. After sleep deprivation in mice, slow wave activity and the duration of non-REM sleep increase, so this is used to measure sleep need in mice. The researchers who did this study used sleep deprived mice, as well as the Sleepy mutant mouse model (I’m not being cute, this is the actual name of the mutant strain).

The Sleepy mice have a mutation in a gene called Sik3 that encodes for an enzyme. The mutation causes the enzyme to work more efficiently and the mice sleep more, but have an elevated need for sleep (as measured by the brain waves). So these mice are always tired due to one amino acid change in one enzyme – that’s powerful.

Phosphorylated proteins drive sleep need

The researchers compared normal mice with the sleep deprived and Sleepy mice, looking at the chemical changes to the proteins in their brains. The sleep deprived and Sleepy mice had more phosphorylated proteins than the mice who had a normal amount of sleep.

Phosphorylated? That’s a mouth full (here’s how to say it). There is a small molecule called a phosphate, made up of a phosphorous atom surrounded by oxygens. This chemical group is big and charged and will change the shape of the rest of the protein when it is added on. Since phosphorylation changes the shape of proteins, that may also change the way the proteins function.

Phosphorylation changes the shape of the protein (from Campbell's "Biology")

The longer the sleep deprived mice stay awake, the more phosphorylated proteins there are. If the mice are allowed to sleep after being deprived, their proteins go back to the unphosphorylated state.  

Sleepy mice are always in need of sleep, regardless of how much sleep they get, so their proteins are always phosphorylated. Why do Sleepy mice have so many phosphorylated proteins? Remember that the Sleepy mice have a mutation that makes the Sik3 enzyme more active. Guess what the function of Sik3 is! It is a kinase enzyme, which adds phosphates to proteins. So the poor Sleepy mice accumulate phosphorylation at a higher rate than normal mice, so they will always have an increased need for sleep.

Many of the proteins that are being phosphorylated during the awake state function at the synapse, where neurons communicate with each other. Some neuroscientists believe that memories are encoded while we are awake by changes to synaptic function. These synaptic changes are refined during sleep to consolidate the memories in long-term storage. The authors suggest that the accumulating phosphorylation regulates synapse function and memory formation, though they don’t show evidence for the connection with memory.

In conclusion, next time you are getting tired at that concert, just tell your friends, “My synaptic proteins are too phosphorylated, I need to go home.” They’ll understand.

Videos!

Cellways, it's been awhile! Here are a couple of videos I made in the last years:


A Ted-Ed video about X-chromosome inactivation and some interesting consequences of that.

A Science Sketches video about the basics of stem cell biology.

Microbiome accelerates neurodegeneration

Parkinson disease (PD) is a neurodegenerative disease characterized by motor deficits and aggregates of a protein called α-synuclein (α-syn) in the brain (pronounced sin-NU-clee-in). Genetics plays a role in PD, because there are some early-onset forms of PD that are caused by mutations in α-syn that cause it to more readily clump together and form the protein aggregates. The purely genetic forms of the disease, though, are relatively rare, so the environment must also play a role in most cases. A recent paper published in Cell by Sampson et al. explores how the microbiome in the gut affects development of PD symptoms.

The microbiome is the community of bacteria and fungi living in and on us (watch this awesome video about the microbiome). It has previously been shown that the normal gut microbiome is disrupted in various diseases such as autism and in Parkinson’s patients. It’s always hard to know, though, what is the cause and what is the effect. Does the disease cause the microbiome to change, or does the change in the microbiome cause the disease? Maybe a little of both.

Mouse model

To address the role of the microbiome in Parksinson Disease, the authors relied on an established mouse model of PD. These mice overexpress the normal human form of α-syn in all their neurons. Even though this isn’t the mutant form of the gene, the fact that it is overexpressed all over the brain causes the characteristic α-syn aggregates. These mice are slow in motor tasks, including removing a piece of tape from their noses (sounds like a frustrating, but also adorable behavioral task). They also have impaired gastrointestinal function, which is to say they don’t produce as much poo as other mice. [An aside: normal mice apparently drop about 7 fecal pellets every 15 minutes!]

The researchers took these mice with mouse-Parkinson’s and raised half of them in a super sterile environment where they have no microbiome (called “germ free” mice), and the other half got all dirty so they had a microbiome (I will call these “dirty mice”). The PD mice with a microbiome had way more motor impairments than the mice without a microbiome! Yes, I wrote that correctly. I thought the microbiome was supposed to help its host? Well, not in these mice overexpressing α-syn.

Get this: if you give the dirty mice antibiotics from age 5-13 weeks old and then test them, they were more like the germ free mice – no motor impairments and better fecal output. Not that you would want to give humans antibiotics for their entire lives (that could cause some autoimmune diseases and serious digestive issues), but this does demonstrate that it is the gut microbiome that is affecting the symptoms of Parkinson Disease.

Short chain fatty acids

The bacteria living in our gut produce all sorts of chemicals that can get into our blood and nervous system. Bacteria produce short-chain fatty acids (SCFA), which are basically just little fats that can cross over the intestinal lining and get into our bodies. Parkinson’s patients produce more SCFAs, so the authors tested the role of SCFAs in their mouse model.

Germ free mice overexpressing α-syn are relatively normal, right? The authors fed these mice a bunch of SCFAs to mimic what the gut bacteria would be making and the mice became impaired like the dirty mice (can’t get that tape off their nose). This is amazing to me. So short-chain fatty acids that are normally made by the gut bacteria are sufficient to cause the Parkinson’s symptoms. Note that feeding SCFAs to normal mice without all that α-syn did not cause Parkinson's symptoms.

           α-syn mice no microbiome + SCFAs = impairments of α-syn mice with microbiome

Microbiome and the immune system

What are the short-chain fatty acids doing to the nervous system? One important role of the microbiome is to train the host’s immune system so it knows what to attack and what to ignore. This is why the microbiome plays a role in the development of autoimmune diseases, where the body attacks the wrong things (like a harmless pollen molecule or the body’s own cells like in type I diabetes). SCFAs can get up into the brain and regulate the immune cells of the nervous system, called the microglia (pronounced micro-GLEE-a). Indeed, the dirty mice with a full microbiome had more activated microglia in the brain than the germ free mice. Likewise, the germ free mice fed SCFAs also had activated microglia.

An overactive immune system promotes protein aggregation, so here’s the model: something causes the microbiome to become unhealthy, which causes the release of a lot of SCFAs, which activate the immune system in the brain, leading to neuron death and protein aggregation. The diagram below has some extra information in it, but the pathway in black is what they showed in this paper.

What about human patients?

Okay, so the microbiome plays a role in this one particular mouse model of PD, but what about in humans? Remember that the microbiome and the amount of short-chain fatty acids in Parkinson’s patients are different than in healthy humans. The authors took the microbes from human feces and transplanted it into the guts of the germ free α-syn mice. Amazingly, the germ free mice that got the bacteria from Parkinson’s patients had more severe motor impairments than the mice that got bacteria from the healthy humans. So there’s something going on in the microbiome of humans with PD that enhances the symptoms.

The authors raise the point that two things were needed for these mice to have the symptoms of Parkinson disease:

1) Overexpression of α-syn (genetics)

2) Disordered microbiome, also known as dysbiosis (environment)

This is a great example of a complex disease that is caused by the interplay of genetics and environment. Perhaps this information can be used to come up with new treatments to correct the dysbiosis and slow down the progression of Parkinson disease. 

Human language in dog brains

Spoken language conveys meaning in two ways: the meaning of the words (semantics or lexical knowledge) and the intonation that the speaker uses. We can sense questions by the rising pitch at the end of the sentence. Likewise, we can tell if someone is upset or being sarcastic based on how they say the words. The patterns of intonation in language is known as prosody. There are areas of the brain that are specialized for decoding the semantic meaning of language and different areas for interpreting prosody. In fact, you can have damage to one area during a stroke, while the other area remains intact. There are great examples of this in “The President’s Speech” in Oliver Sacks' book The Man who Mistook his Wife for a Hat.

In most people, word meanings are processed by the left side of the brain and prosody is localized to the right side of the brain. Some animals also use the left side of their brains to understand meaningful and familiar sounds of their species (like alert calls or bird songs). What about for animals, like dogs, which can understand the sounds of another species (i.e. commands from humans). Is the dog brain really processing the intonations of praise “good dog!” or are they responding to the words? Do they process meaning and intonation separately like humans do?

Dogs in MRI machines

In the latest issue of Science, Andies et al. published their studies of language processing in dog brains. My first thought when I read the abstract was “how do you get a dog into an MRI machine?” We commonly study which areas of human brains are active during different tasks using a technique called functional MRI (or fMRI). fMRI was done on these dogs while they listened to their trainers speak. If you have ever had an MRI scan, you know they strap you in and you cannot move your head at all. Same thing with these dogs. Needless to say, they were very well trained dogs. If you still can’t believe it, check out this video the researchers made and the cute photo of dogs in an MRI machine below.

Really well trained dogs lying still before their MRIs. (Image from phys.org)

Dogs process language like humans

Okay, so they got the dogs in the MRI machine and scanned their brains while they heard their trainer say different things. The trainer would either say words of praise, like “good boy” (in Hungarian), or neutral words. And they used either a neutral, flat intonation or they raised the pitch of their voice to create a praising intonation. This created four possibilities:

• Praise words with praising intonation
• Praise words with neutral intonation
• Neutral words with praising intonation
• Neutral words with neutral intonation

They compared the brain responses to each combination and found that the left side of the brain responded to words of praise regardless of the intonation. This is amazing, right? The dogs have heard “good boy” enough times that their brains responded specifically to that phrase regardless of how it was said. It’s like they sort of know what it means. It would be interesting to see if they respond to the same phrase spoken by a stranger.

The researchers also found that the right side of the brain had active areas when praising intonation was used, regardless of the word meaning. So dogs also understand how our voices change when we praise them.

Finally, the researchers looked at areas of the brain associated with reward. These areas are active in a variety of animals when they receive natural rewards like food or during sex, but the reward pathways are also active if the animal is given an addictive drug like cocaine. Alternatively, you can put an electrode into a mouse brain that stimulates the reward pathway and the mouse will push a lever to receive an electrical shock in this area of the brain over and over until it starves.

Andies et al. found that praising words spoken in a praising intonation activated the reward pathway in the dogs. Praise words alone and praise intonation alone had no effect. So dogs really do feel good when you say “good dog” in a high pitched voice.

Notice the organization of language processing in the dog brain. Just like in humans, language semantics (praise vs neutral words) was processed on the left side and prosody (praise vs neutral intonation) was processed on the right side. What does this tell us about the evolution of language? Language lateralization has likely been around a long time and is not uniquely human. The authors end the article with this gem: “What makes lexical items uniquely human is thus not the neural capacity to process them, but the invention of using them.”

No Y genes? No problem, bro.

The Y-chromosome is one of the smallest chromosomes in the human genome and contains genes involved in male development and production of sperm. Previous research has shown that just two genes on the Y chromosome are necessary to make male mice who can sort of produce sperm. By “sort of” I mean that the mice make things called “round spermatids”, which genetically are the same as sperm, but are underdeveloped, so they can’t naturally fertilize an egg. A lab in Hawaii took these round spermatids and injected them into oocytes to demonstrate that the resulting zygotes are viable and develop into normal mice. In other words, the experimental mice have only one X chromosome and the two Y genes, and they develop into males who can reproduce with a little help from scientists.  That is pretty amazing that only two genes can make a male.

The necessary Y genes

So what are these two genes? One of them is called Sry, which encodes for a transcription factor that regulates expression of other genes important for the development of the male reproductive system (see the figure below). The other necessary gene is Eif2s3y, which is involved in protein synthesis and somehow necessary for the production of sperm. There is a similar gene on the X-chromosome, which may serve the same function. Normal XY males express both Eif2s3y and Eif2s3x, the version on the X-chromosome.

 

In a paper that came out earlier this year in Science, Yamauchi et al. asked whether they could replace the function of Sry and Eif2s3y with other genes that are found on other chromosomes. Instead of a male mouse with Eif2s3y, what if you made a mouse that was overexpressing Eif2s3x?  Could the X version compensate for the Y version? And instead of Sry, could you overexpress one of its target genes to replace its function?

Through the power of mouse genetics, the researchers created a mouse line with one X-chromosome and no Y-chromosome, which overexpressed Eif2s3x and Sox9, one of the Sry targets. In other words, these mice do not have any genes that are normally found on the Y-chromosome.

A male mouse with no Y

The mice with no Y-chromosomes and no Y genes, but overexpression of Sox9, developed into males, with male reproductive systems (though smaller and less developed). When Eif2s3x was overexpressed along with Sox9, the males were able to produce the round spermatids (precursors for sperm). The researchers did their artificial fertilization with these round spermatids and were able to produce healthy offspring. 

 

So just to repeat: the mice without a single gene from the Y-chromosome developed into males and produced sperm that are good enough for successful in vitro fertilization. Just by overexpressing two genes found on other chromosomes. That’s amazing!

Pandas are lazy!

Pandas are closely related to carnivorous mammals (like all the other bears), but they consume mostly bamboo.  Their digestive tracts are short and adapted for digesting meat, not cellulose that is found in plants.  In fact, they only digest about 20% of all the bamboo they eat, and they eat a lot of bamboo (30-60 pounds a day)!  How are these large, adorable bears able to get enough energy to function from their inefficient digestion of bamboo?  Researchers in China and Scotland addressed this question by studying captive and wild pandas, described in a recent Science article.

Low energy expenditure

Nie et al. measured the daily energy expenditure of the pandas and found that they used an unusually low amount of energy, only 37.7% of the predicted value based on their body mass.  In fact, pandas are expending energy at levels similar to the three-toed sloth, the epitome of a low-energy mammal.  The measly amount of nutrients they get from all that bamboo would be able to sustain such a low energy expenditure, so that’s how the panda is able to get by with such a maladapted digestive system.

How do the pandas manage to spend so little energy?  There must be some adaptations that are allowing the panda to survive without expending so much energy.  The authors found a number of these adaptations:

1) Pandas have a thick layer of fur, so they can maintain their internal body temperature with less heat loss through the skin.  The researchers measured temperature at the surface of various animals and the pandas consistently were cooler than other mammals (like a cow or dog).  Their internal body temperature would be considerably warmer because the fur helps insulate them, so they don’t have to spend as much energy on maintaining their body temperature.

2) Pandas are lazy.  No surprise: pandas spend more time inactive and when they do move, it is slowly.  So that is less energy needed for muscle contractions.

3) Pandas have small brains, livers and kidneys, so their organs need less energy.

4) Pandas have a low resting metabolic rate, which is driven by the thyroid hormones, T3 and T4.  In fact, levels of these two hormones were considerably lower than for other mammals of the same body mass, even lower than a hibernating bear.  The thyroid hormones regulate protein, carbohydrate and fat metabolism, as well as growth and development.  If the pandas don’t need to produce as much heat or energy, then there is no reason to have a high metabolic rate.

Interestingly, pandas have a single mutation in a gene called DUOX2, which is not found in any other mammals.  DUOX2 encodes for a protein that is necessary for the production of T3 and T4.  The mutation causes a premature “stop” in the protein, so it likely affects the function of DUOX2. 

In other words, pandas cannot synthesize T3 and T4 as well because of this mutation, so they have a reduced metabolic rate.  But that’s okay, because they are good at maintaining their body temperature and they have developed an enjoyable lifestyle of relaxing and eating.  The fact that their digestive tracts have not evolved for plant digestion is alright given the fact that they don’t really need that much energy from their food.  So it all works out: pandas are able to survive on their diet of bamboo and we can watch them sit around.

Bigger brains with Frizzled HARE

We have all heard that the sequence of human DNA differs from chimpanzee DNA by only about 1%.  Yet humans are capable of building complex civilizations while the chimps are still eating bugs in the forest.  If you compare the human brain to the brain of any other primate, it’s easy to see where our sophisticated cognitive abilities come from. 

From thebrain.mcgill.ca

DNA is the blueprint for making proteins, cells and organs, so is there something special hidden in that 1% sequence difference that gives humans bigger brains?  In particular, scientists have focused on regions in the human genome that have undergone rapid sequence changes in the human lineage, but not in other primates.  Besides looking for differences in genes that make proteins, we can also look for changes in regulatory regions, like enhancers, that control when and where the genes are expressed.

A recent paper in Current Biology by Boyd et al. explores these questions by studying a human-accelerated regulatory enhancer (HARE5), which differs significantly between humans and chimps.

Enhancer activity of HARE5

How do you study enhancers?  One way is to use a reporter gene.  Enhancers drive expression of nearby genes, so what if you swapped out a nearby gene and replaced it with a gene for a fluorescent protein?  Then you can look at your organism and wherever you see the fluorescent protein, the enhancer is active, meaning that the normal “nearby gene” is normally expressed in those cells.  Instead of doing these experiments with chimps and humans, which would take forever and be unethical in some cases, the authors put these reporter constructs into mice.  The enhancers from the chimps and humans drove expression of the reporter gene in the embryonic mouse brains.  The gene adjacent to the human enhancer was expressed earlier in development and more strongly than when placed next to the chimp enhancer (in other words, a lot more protein is being made).

Reporter gene experiment.  The mouse brain images are actual results from Figure 2 in Boyd et al. (2015).

This tells us that whatever normal gene is near HARE5, it is probably expressed earlier and way more in humans than in chimps.  There are just 10 sequence differences in the human HARE5 (i.e. mutations), which is enough to affect the way the enhancer functions and activates expression of genes. 

Frizzled expression is regulated by HARE5

So which genes are near the HARE5 sequence?  The closest gene is called Frizzled 8 and it is a receptor that responds to signals sent by other cells.  Frizzled 8 (FZD8) is a well known component of the Wnt signaling pathway that regulates many aspects of embryonic development, including neurogenesis (formation of new neurons).  The authors demonstrate that the mouse HARE5 physically interacts with Fzd8, which is a necessary  first step of gene expression, so Fzd8 is likely affected by the HARE5 sequence differences in humans and chimps.

The authors wanted to see what would happen to development of the mouse brain when Fzd8 is expressed in the same pattern as in humans or chimps.  They repeated the earlier experiments, but this time instead of using a reporter gene, they put the mouse Fzd8 gene next to the chimp or human HARE5 sequence.  They injected these DNA constructs into mice and waited to see what would happen to embryonic brain development.  When the chimp-HARE5 was driving expression of Fzd8, not much changed in terms of mouse brain development.  However, when the human-HARE5 sequence was activating the mouse Fzd8 gene, the mouse brain grew 12% bigger!! 

Let me be clear here-- they are not expressing the human Fzd gene in mice.  No, they are using the human enhancer to drive expression of the mouse Fzd8 gene, so presumably it is expressed more and earlier in development (like they saw in the reporter gene experiment).  The neural progenitor cells (pre-neurons) divided faster than in a normal mouse, leading to formation of more neurons, and a bigger brain! 

10 sequence changes in an enhancer may be one reason why I am able to write and you are able to read and understand this blog.  Whoa.  No news yet about whether these mice with bigger brains are also able to read and write… I’m sure they’re saving that for another paper.

I should say too, that there are probably a number of other similar changes to other enhancers and genes that all led to the rapid development of the big ol’ human brain.

Here's another blogger's take on this paper 

Smart phone use changes the brain

One of the most remarkable things about our brains is how organized they are. Sensory information from our eyes, mouth, skin, nose and ears goes to different locations in the brain. For example, visual signals are processed first in the very back of the brain, whereas sensations of touch and pain activate the middle region of the brain called the somatosensory cortex.

Functional organization of brain cortex. (Source: imgarcade.com/1/sensorycortex)

Remarkably, the brain gets even more organized from there. Within the visual cortex, there are columns of neurons that only respond to light that is horizontal and others that only respond to lines that are tilted 45 degrees. The somatosensory cortex is also highly organized, with different parts of the body represented by specific sets of neurons. If you were to send electrical shocks into one specific area of the somatosensory cortex to activate those neurons, you may elicit feelings of touch from the right thumb, even though the subject is not being touched at all. Move those electrical signals over slightly to another area, and the subject may feel touch instead coming from the palm of their hand.

Wow, right? But here’s the real mind blower: this organization can change over time as the person experiences different sensory inputs. If you are a violin player, you feel the strings with your fingertips a lot, so the fingertip part of the somatosensory cortex is super active. This extra activity allows the fingertip representation in the brain to grow and recruit nearby neurons to also respond to touch in the fingertips. The cortical representations are “plastic” and always changing with use.

A violin player may practice this one particular skill a lot, but what about other activities we do everyday with less intensity, like using smart phones? Think about how often you are swiping the screen with your thumb. That’s a lot of sensory information being sent to the thumb part of your somatosensory cortex. Would this increase the thumb representation in your brain? A recent paper by Gindrat et al. addressed this exact question using EEG to record brain activity in smart phone users versus people with the old-style cell phones.

Electroencephalography

How can you actually measure the area of body representations in the somatosensory cortex? You could stick electrodes into people’s brains and record the activity in their neurons, but that’s a little invasive. You could put them into a MRI machine and measure brain activity when you touch their thumbs, but that is time consuming for so many subjects (37 total). Instead, the authors used a method known as electroencephalography, or EEG, which consists of 62 surface electrodes placed on the scalps of the subjects. Each electrode records the summed electrical activity from all the neurons positioned right under the electrode. Before an experiment, all the electrodes would be picking up a baseline of activity from lots of different neurons firing asynchronously. However, during an experiment, there is a single stimulus (like touching the subject’s thumb), which elicits activity in a lot of neurons all at the same time. This activity summates to give one large response called the event related potential (ERP), which is recorded by the nearest electrodes.

EEG electrodes record brain activity (source: Wikimedia commons)

Finger representations in smart phone users

The ERPs for the thumb, index finger and middle finger were larger for the smart phone users than for the non-touchscreen users. There was a correlation between the amount of phone use per hour and the ERP, so the more use, the greater the ERP, which is to say the more activity in the somatosensory cortex. The number of electrodes recording the ERP was greater in the touchscreen users, so when you touch the thumb of a touchscreen user, a larger part of the somatosensory cortex responds. In other words, the thumb representation was larger in smartphone users who use their thumbs more often.

The more recently the subjects had used their phones intensely, the larger the ERP for the thumb, which indicates that brain remodeling occurs on a very short time scale (within 10 days in this experiment). Interestingly, there was no correlation between ERPs and the age at which the subject started using a touchscreen. This is in contrast to the previous experiments done with trained violin players, which did show a correlation between the size of the finger representations and the age at which they first started playing. The authors suspect that a trained violinist develops a more stable sensory representation than touchscreen users who are casually using their phones (as opposed to years of disciplined practice).

So the take-home message is that normal day-to-day activities can influence brain plasticity and the way our sensory representations are organized in our brains. This could be a good thing, because subjects develop better touchscreen skills. On the other hand, the enlarged thumb representation could cause focal dystonia, which is characterized by involuntary muscle contractions and sometimes pain, as the various body part representations lose their distinct boundaries and start to overlap. This probably won’t be a problem for most phone users, but be forewarned all you smart phone addicts out there.

What big nuclei you have!

Eggs get ready for fertilization by producing and storing all the proteins necessary for early embryo development.  After fertilization, there are a series of rapid cell divisions without growth, producing a lot of small cells (here's a video).  At some point during this process, the embryo switches over from using the proteins from mom, to expressing their genome to make their own proteins.  This transition to embryonic transcription is known as the midblastula transition, or MBT.  How does the embryo know when it is time to turn on gene expression? 

One theory is that the ratio of nuclear to cytoplasmic volume (N/C volume) is the trigger for MBT.  The nucleus is where DNA is stored within a cell; this is where gene expression occurs.  The cytoplasm is the goo that the nucleus sits in.  During those rapid early cell divisions, nucleus size does not change much, while the cytoplasm in each cell keeps getting smaller and smaller.  The N/C volume increases, since the cytoplasmic volume is decreasing.  Is there a certain threshold of N/C volume, above which the embryo switches on gene expression?

Jevtic and Levy did a series of clever experiments, using frog embryos to address this question, which was published today in Current Biology.  In Xenopus laevis frogs, the midblastula transition always occurs after the 12^th cell division.  The researchers manipulated the nucleus size in the frog embryos to see if that would change the timing of MBT.

Changing nuclear volume

The authors were able to increase nucleus volume by injecting embryos with mRNA for importin and a type of lamin.  Importin acts as a shuttle that brings other proteins into the nucleus, including structural proteins that make up the nuclear envelope.  Lamins form the inside of the nuclear envelope, so by injecting the mRNA for these two proteins, they caused overexpression of nuclear proteins that will make the nucleus grow larger.  To decrease nuclear size, they instead injected mRNA for a protein that causes another cell structure to grow (the ER) at the expense of the nucleus.

They injected the mRNAs and a red dye into one cell in the two-cell stage.  When this cell divides, its daughter cells inherit the red dye and the mRNAs and proteins that change the nucleus size.  Thus, by the time a normal embryo is ready to undergo MBT (the midblastula transition to express their own genes), half of it will be red and have abnormally sized nuclei and the other half will be normal and act as an internal control. 

N/C volume triggers MBT

They looked at embryonic gene expression (as a readout of MBT) in the cells with abnormal nuclei at different developmental stages.  The cells that had larger nuclei reached the critical nucleus to cytoplasm (N/C) ratio earlier in development and began expressing embryonic genes earlier than the neighboring cells with normal nuclei.  Likewise, the cells with smaller nuclei took a little bit longer than normal to undergo MBT.  I love that the two halves of the embryo are out of sync with each other just because the sizes of the nuclei are different.  This really shows that there is a critical N/C volume and manipulating this ratio is sufficient to initiate the midblastula transition.  

How do the cells know the size of the nucleus and cytoplasm?  The authors suggest that the oocyte must have inhibitors in it that repress transcription, so the embryo’s genome is inhibited at first.  As the cells divide, these inhibitors are split among them, so the inhibitors become less and less concentrated in each cell.  Once they reach a certain low concentration, they no longer function, so the cells can begin transcription.  This would explain why increasing nucleus size would cause an earlier midblastula transition: the larger nuclear volume essentially dilutes the inhibitor further, so it reaches that low threshold concentration sooner.  It’s important to get the timing of gene expression just right during development and the N/C volume appears to be one way that cells manage to do this.

Improving reproductive cloning

Remember when Dolly the sheep was cloned in 1996?  That was the first cloned mammal and everyone freaked out thinking we would be cloning all our pets and even humans within a few years.  Well, nearly 20 years have passed since then and reproductive cloning is still a very difficult and inefficient procedure.  Most cloning has a 1-5% success rate.  Why is that?  Before we can answer that, we need to understand the procedure for reproductive cloning.

Somatic cell nuclear transfer

Our bodies are made up lots of different types of cells – neurons, skeletal muscle, intestinal cells, immune cells, etc.  Despite the different functions and structures of these cells, all of the cells in one organism have the same genome, the same set of genes.  What makes cells unique is that they express different genes at different times, so different proteins are made.  What this means is that the blueprint (DNA) for making a new organism is right there in every cell in your body.

The normal way of making an embryo is by taking half of the genome from a male (in sperm) and half from a female (in the egg) and combining them during fertilization.  In reproductive cloning, you already have a whole genome from any adult cell.  That nucleus from the adult can be inserted into an oocyte (or egg) that has had its DNA removed (“enucleated”).  The egg is necessary because it has lots of nutrients and signals in it that are important for the first few cell divisions during early development.

This process is shown in the diagram below and is called somatic cell nuclear transfer.  In the example, the adult genome is coming from a fibroblast cell and is transferred into an enucleated oocyte.  This is a way to get stem cells (ntES), which can be used for therapeutic purposes, like making more neurons that can be transplanted into someone with Parkinson’s.  Or you could let the cloned cells grow up into an embryo and then into a cloned organism.

Somatic nuclear cell transfer (from Stembooks.org)

Although it is possible to make cloned organisms using adult donor genomes, the efficiency is much higher when using genomes from embryos.  What happens to the adult genome that prevents it from directing the formation of a new organism?  This problem is addressed in a recent paper by Matoba et al., published in Cell.

Epigenetic changes

Although adult cells should have the same DNA sequences as their embryonic precursors, the genome can be organized differently, which can affect which genes are expressed.  DNA wraps around histone proteins as a way to organize the long DNA chains.  Histones can be modified in such a way that the DNA will wrap around more tightly or more loosely.  For example, if a particular amino acid in histone 3 is trimethylated (three CH3 groups are added), then that makes the DNA pack up closer together, so it is really hard to express those genes.  There are genes that may need to be expressed early on in development, so their histones will be modified to allow for loose packing, but then after they are expressed, they’ll get packed away, so they take up less space.  These kinds of modifications that affect gene expression are called epigenetics.

As an analogy, imagine you have had a child, so you have baby clothes, a crib, car seat and toys in your house.  Once that child grows up, you take all those baby things down to the basement.  You still have them, but you will probably never need to use them again, so you can pack them all up and store them so they are out of the way.  It may be hard to access them again, but they are still there.  So a gene that has been packed away into condensed chromatin is still present in a cell, but it is no longer giving instructions for making proteins, unless something comes along and unpacks it.

You can see now the problem with somatic cell nuclear cloning.  The adult cell already has some DNA packed away, so when the genome is put into an oocyte, it may be impossible to express the genes necessary to direct normal development.  In the paper by Matoba et al., they did indeed find that there are more trimethyl modifications (called H3K9me3) in mouse embryos derived from nuclear transfer than embryos from in vitro fertilization (using a sperm and egg).  These regions were associated with decreased gene expression and compact DNA.

Improving efficiency

Now we know one of the problems, but what can researchers do to improve the efficiency of reproductive cloning?  Somehow they need to decrease H3K9me3 modifications in the donor genome.  They do this two ways:

(1) There are too many methylated histones, so the authors injected an enzyme that removes methyl groups into the one-cell embryos.  The embryos expressed more genes and survived throughout development.  70% of these cloned embryos implanted into a surrogate mouse uterus and 8% survived to adulthood.  Those numbers are higher than before, but still not perfect.

(2) They also tried decreasing expression of the enzymes that put on the methyl groups.  This also improved development, so 50% of the embryos made it to later stages of development, but they did not see how many survived to adulthood.

There is an epigenetic barrier for nuclear transfer from adult cells in mouse oocytes.  Presumably a similar problem is preventing cloning in other organisms as well.  It makes sense that an adult cell would have a different pattern of epigenetic modifications than an embryonic genome.  The authors were able to improve cloning efficiency by decreasing the H3K9me3 modification, but there are probably other histone modifications that are also different in adults.  There is still a long way to go before cloning is a reliable procedure, but at least now we have some explanation of why it is so difficult.

Transgenerational inheritance of fear

A new semester has begun and I have no extra time to update this blog, so just a short entry today.  This paper was just too cool to pass up.  It was published earlier this year in Nature Neuroscience by Dias and Ressler.  They conducted a series of experiments which showed that learned fear can be passed on from generation to generation in the sperm DNA.  That’s Lamarckian evolution for all you evolution nerds out there.  A learned behavior that is inherited genetically -- totally crazy!

The DNA sequence itself isn’t changing, but instead the expression of genes is altered, so different amounts of proteins are being made.  This process is known as epigenetics (which I’ve discussed before with regard to histone modifications).  One way to change DNA expression is by methylating cytosines (the “C” in DNA sequences).  The methyl group (CH3) makes it harder for proteins to bind to the DNA and transcribe the genes into mRNA and subsequently into protein.  The general rule of thumb is: more methylation --> less gene expression, less methylation --> more gene expression.  This is a common way our cells regulate gene expression, and what’s really interesting is that many external influences can affect DNA methylation, like traumatic life experiences, smoking, exercise, environmental toxins, etc. 

Cytosine getting methylated (note the H3C addition on the the molecule on the right)

It is conceivable that DNA methylation patterns can be inherited through generations, so changes in gene expression that affected your great grandparents could still be maintained in your cells.  Most of these types of studies focus on changes that occur to the mom during pregnancy.  For example, lets say that researchers expose a pregnant mouse to a toxin that may affect DNA methylation.  The next generation (the F1 generation) is also being exposed in utero to the same toxin.  The eggs or sperm progenitor cells are also developing in the embryo, so the next next generation (F2) may also be exposed to the toxin.  Thus, if you see DNA methylation changes in the F1 and F2 offspring, this isn’t really so surprising since these cells were all exposed at the same time as the mom (F0 generation).  In order to really prove that DNA methylation patterns can be inherited across generations (transgenerationallly), you need to expose the parents to a stimulus before conception, before the F1 and F2 generation even exist, which is what they did in this paper.

The authors chose to initiate changes to gene expression by conducting odor conditioning in mice.  They paired a particular odor with a mild foot shock and conditioned F0 males to be afraid of the odor.  Then these males mated with naïve females (never exposed to the odor).  The F1 offspring showed excessive fear to the conditioned odor, even though they had never encountered it before.  It was their fathers who had been shocked, not them.  The authors found there were more cells in the olfactory region of their brains that expressed the olfactory receptor for the conditioned odor.  Furthermore, their dad’s sperm and their own sperm were less methylated in the gene that encodes for that particular odor receptor, so the offspring of F1 were also affected.

F0 dad learns fear for odor --> decreased methylation for receptor gene --> F1 offspring inherit methylation changes --> express more of the receptor --> increased sensitivity to odor --> F1 sperm have same decreased methylation --> sensitivity for odor passed on to F2 generation  

Grandpa (F0) had a bad experience with an odor and now his grandkids will be more afraid and sensitive to that odor.  And it’s all genetic.  The authors did a series of experiments to show that it isn’t behaviorally based (grandpa isn’t telling the grandkids about his horrible experience with this smell).  For instance, they took the sperm from the F0 mice and took it to a different mouse facility and did in vitro fertilization, so the father was nowhere near his offspring or the mother.  The F1 mice from in vitro fertilization were just as super sensitive to the odor.

This is nuts!

Here's a good blog post about this paper