The mosaic female brain

Female mammals have two copies of the X chromosome while males have only one copy (because they have a Y chromosome instead).  Chromosomes contain genes and genes are the instructions for making proteins, so if females have twice as many copies of each gene on the X chromosome, will they make twice as much protein?  The answer to that is mostly “no”.  In young female embryos, one X chromosome is randomly inactivated and will remain that way through her life.  The chromosome gets compacted into a structure known as a Barr body.  However, when X inactivation occurs there are many embryonic cells and each one can inactivate one copy or the other.  Why does this matter?  Well, remember that one X chromosome came from dad and one from mom, so there may be different variants for each gene; different versions of proteins can be made depending on which X chromosome is still active in that cell.  In other words, females are genetic mosaics, where each cell may express one X chromosome or the other.  That’s cool!

What if the female embryo inherits one good copy of a gene and one bad copy that is non-functional and disease-causing?  Some of her cells would express the good copy of the gene and be fine and other cells would express the bad copy and be messed up.  The severity of the disease for this female will depend on how many cells inactivated the good copy and where these cells are located in the body.  Imagine that X inactivation occurred at the 4 cell stage, where two cells inactivate the good chromosome and the other two cells inactivate the bad chromosome.  Once an X chromosome is inactivated, it will stay that way in all the cells that are formed from that original cell in the 4 cell stage (see the figure below).  If each one of those 4 cells divides the same amount to form the final adult form, then you would expect half of her cells to be messed up and half of them to be fine.  But what if the two cells with the active bad chromosome happen to be cells that will divide way more and make way more future tissues of the body?  Then in the adult form, she would have tons of messed up cells and probably have a much more severe version of the disease.

In females one X chromosome is inactivated early in development (image from www.scoop.it)

As I mentioned earlier, X inactivation actually happens later on in embryonic development when there are more cells and each one can choose to inactivate one chromosome or the other.  If we consider the disease scenario, this random nature of X inactivation can lead to huge variability in X-linked disease expression in females.  It’s also important to think about how certain types of cells and tissues develop.  If an entire tissue develops from a single cell after X inactivation, then all of the cells in that tissue will have the same inactivated chromosome. 

Researchers at John Hopkins University visualized X inactivation by marking expression from each X chromosome with a different fluorescent protein.  Wu et al published their beautiful images in a recent article in the journal Neuron.

Marking X chromosomes

The authors created two types of mice, which each had an extra inserted gene on the X chromosome.  One type had a gene that encodes a red fluorescent protein called tdTomato.  The other mice had a gene for the green fluorescent protein, or GFP, which was originally discovered in jellyfish.  They then mated these two mice together and used the female offspring that had one X with tdTomato (X_t) and one X with GFP (X_G).  If the “red” chromosome is inactivated, then only GFP will be expressed and this cell will look green, as will all of its daughter cells.  This way they can look at the heterogeneity of X chromosome expression in different parts of the body. 

The results

Overall, the mice came in all different amounts of red and green.  For instance one mouse might be nearly all green while its sibling is all red, again indicating that X inactivation is a random process.  In the mice that had both red and green, it was interesting to see the different patterns in the body.  For instance, in the intestine, cells of the same color were found in columns.  That’s because the cells in the column originate from one single stem cell, so they should all contain the same active X chromosome.

X inactivation appears in columns in intestinal tissue, because cells from a single stem cell migrate together

 Another interesting finding was that skeletal muscle cells expressed both red and green fluorescent proteins.  This would seem to indicate that there is no X inactivation in muscle, but this is not the case.  Skeletal muscles are actually formed by muscle progenitor cells (myoblasts) that fuse together, creating cells with multiple nuclei and copies of the genome.  If a cell with an active “green” X chromosome and a cell with an active “red” chromosome fuse together, then the muscle will express both proteins.  This only works for skeletal muscle; cardiac muscle in the heart does not develop by cell fusion, so these muscle cells are either red or green.  This is a great demonstration of the differences in muscle development.

Skeletal muscle cells express both X chromosomes, because they are formed via cell fusion

 They also noticed clear differences between the left and right side of the body, like in the tongue, retinas and brain.  This indicates that progenitor cells stay segregated to either the left or right side during development.  In other words, there is not a lot of migration between the two sides of the body, where a cell on the right side would make cells for the left side of the body, and vice versa.

The mosaic brain

The main focus of this paper is on the heterogeneity in the nervous system.  They looked at two different cell types in the brain: excitatory pyramidal cells and inhibitory interneurons.  These two types of neurons develop from different areas of the embryonic brain.  They found that inhibitory interneurons were highly mixed.  When they quantified the fraction of red inhibitory cells in two different parts of the brain, the values were very similar.  On the other hand, when looking at excitatory neurons, there was a lot of variability of which X chromosome was inactivated, across different parts of the brain and in different animals.  If there was an X-linked gene that affected excitatory neuron function, then the effects on neuronal circuits would be different for different regions of the brain in an individual.  The authors suggest that this could actually be a good thing, because it would allow females with different genetic variants to respond to a range of stimuli, increasing the dynamic range. 

So there are bad aspects of X chromosome inactivation, like the expression of X-linked diseases, but there are also some good points, like increased functional diversity of neurons.  The authors suggest that X inactivation “may represent one of the more significant mechanisms by which individual differences in central nervous system function are generated.”  It is crazy to think that random inactivation of a chromosome in the early embryo might give us our future individual personalities. 

Release the sperm!

While preparing a class about synthetic biology, I came across this older paper that actually shows a practical application for synthetic biology.  Kemmer et al. describe a new technique for artificial insemination of cows in the Journal of Controlled Release (in 2011).  I’m not condoning these practices in cows; that is a debate for another day.  I am much more interested in the biology behind this ingenious way of improving the timing of artificial insemination.  Let’s get into it.

Luteinizing hormone

Before I describe the synthetic circuits, we have to go over what the luteinizing hormone (LH) does.  LH is released from the pituitary gland in the brain and travels through the blood to the gonads (in males and females).  In females, there is a huge surge of LH release once a month, which triggers the release of an oocyte (an egg) from the mature follicle in the ovaries.  In other words, increased LH causes ovulation.  The LH hormone binds to LH receptors (LHR) which are expressed on the surface of the target cells in the ovary.  When LH binds its receptors, it triggers a molecular cascade inside the target cell, which leads to the production of another molecule called cyclic AMP (cAMP).  cAMP is a versatile molecule that can initiate lots of cellular responses, like changes in gene expression or activation of enzymes.

The current practice in cow farming is to keep an eye on the female cows and when they appear to be in estrus, then the farmers inject sperm into the cow and hope for the best.  Different cows, though, will have different durations of estrus, so it is sort of a guessing game to time the insemination perfectly.  The LH surge regulates release of the oocytes, so what if you could design a synthetic system that also releases sperm in response to LH?  The sperm will be encapsulated and inert until the LH surge initiates the release of the sperm from their holding cell.  The farmer could inseminate the female cow when estrus appears to be close at hand and the female’s own LH will release the sperm at just the right time when the oocyte is naturally released. 

How can the researchers design a holding cell for sperm that is responsive to LH?

The synthetic circuit

The holding cell is going to be a little hollow bead of cellulose (diameter = 350-400 um).  Cellulose is a naturally occurring molecule made up of lots of glucose sugars hooked together.  The cellulose beads will stay intact unless there is an enzyme called cellulase to break all those bonds between the sugars.  The researchers envelop living sperm and modified mammalian cells inside the microbeads and these get injected into the uterus of the female cow.  The sperm seem to be happy inside the cellulose and are still functional when they are later released.

 

The modified cells have two engineered transgenes:

1) We want these cells to be responsive to LH, so the cells must express the LH receptor.  The researchers find that the rat LHR actually works best, so these cells will have the gene for making the rat LHR.

2) Remember that when LH binds to LHR, there will be a rise of cAMP inside the cell.  cAMP will activate a protein called CREB that binds to DNA and activates expression of genes (I’m skipping a few steps here).  Okay, so LH will bind LHR, cAMP levels will increase, CREB will be activated and will bind to specific DNA sequences in front of genes.  The researchers put the cellulase gene right after a CREB binding sequence in the second transgene.  CREB should bind to the DNA and activate expression of the cellulase gene.

Hopefully you can see where this going now.  When LH is released during ovulation, it will also bind to these modified cells and cause expression of cellulase (the enzyme that breaks down cellulose).  The cellulose surrounding the sperm will be destroyed and the sperm will be released at the same time as the egg.  Bam!

The two pathways initiated by the LH surge.  On the left is one of the modified cells inside the cellulose capsule.

Does it work?  The researchers inserted the cellulose implants into the uterus of Swiss dairy cows.  Next they injected the cows with a hormone that triggers release of LH.  The capsules were degraded and sperm released at the same time as the cow naturally released an oocyte.  Fertilization occurred and embryos developed via this well-timed artificial insemination.  The sperm capsules significantly increase the time window for artificial insemination, which takes the guess work out of insemination. 

Look, synthetic biology working in a useful setting, rather than in bacteria or mice.

Probiotics for autism

The human microbiome is a hot topic in biology these days.  It is becoming clear that the microbes living in and on our body can have major consequences for our health and happiness.  In fact, abnormalities in the gut microbiome may underlie one of the great medical mysteries of our time: autism.   That some bacteria in our intestines could affect our behaviors and brain development is mind blowing.

Hsiao et al. recently published a study in the journal Cell that investigated the connection between the gut microbiome and autism using a mouse model of autism.  They were drawn to this subject based on the fact that individuals with autism spectrum disorder (ASD) often have gastrointestinal abnormalities, like irritable bowel syndrome and increased intestine permeability.

Autistic mice?

Apparently you can produce mice that exhibit the “core communicative, social and stereotyped impairments” associated with ASD, by injecting their pregnant mothers with a molecule that stimulates an immune response.  In humans, maternal infection is linked to increased risk of autism in their children.  The production of these mice was the most questionable part of the paper in my opinion.  They never call these mice autistic, and the mice do show impairments associated with neurological diseases.  So perhaps we should think of it as a model of a generic neurological disorder.  For the sake of simplicity, though, I will refer to them as “autistic mice”, but remember that it is not a perfect model system.

They find that the autistic mice have various defects in their gastrointestinal (GI) tract.  For instance, their intestinal walls are leaky, so molecules that are not supposed to be absorbed can cross from the gut into the blood stream.  This problem seems to be caused by the fact that these mice express less of the proteins that make the tight junctions between cells.  Think of these as fences between cells, so molecules can’t sneak through there into the body.  In an ideal situation, all molecules that are absorbed from the gut must go through the cells, a process which is highly regulated. 

Tight junctions prevent molecules from passing from the gut into the blood.  Image adapted from dbriers.com

They find a number of metabolites that are produced in the intestine from bacteria, which end up in the blood of autistic mice, but not in the normal mice.  In other words, these are potentially toxic molecules that they need to get rid of, but the toxins are leaking into the blood of the autistic mice.  That’s not good.  In fact, if you inject one of these molecules into a normal mouse, it will become more anxious, similar to the autistic mice.  They couldn’t reproduce all of the behaviors of the autistic mice just with this one molecule, but it’s a good proof of principle.  Presumably it’s the build up of all of these metabolites in the blood that cause impairments of the nervous system.

Dysbiosis of the intestinal flora

I love that word “dysbiosis”.  It means that the intestinal microbiome is out of whack.  The wrong types of bacteria are in there messing stuff up.  Hsiao et al. found a number of species present in the autistic mice that were not in normal mice and vice versa.  Presumably this imbalance in the microbiome is what is making the gut leaky. 

To prove this, the authors fed the autistic mice a probiotic (a “good” type of bacteria) called Bacteroides fragilis (B. frag).  Interestingly, B. frag never actually colonized the guts of the mice, but just having it pass through helped to restore the normal microbiome.  Some of the species that were only present in autistic mice disappeared after they consumed B. frag.  The leakiness of the gut was almost completely reversed, including expression of tight junction proteins.  It wasn’t a perfect reversal, but a number of those metabolites in the blood decreased back to normal.

Behavior affected by microbiome

To review: when a pregnant mouse has an infection, her offspring show signs of autism (a mouse-version).  Somehow this infection causes the wrong bacteria to colonize the guts of the offspring.  The dysbiosis leads to changes in gene expression and a leaky gut that allows toxic molecules into the blood stream, thus affecting the development of the nervous system.  Consumption of a probiotic at weaning age fixes a lot of the gut issues.  Does it also reverse some of the behavior impairments associated with autism?

The short answer is yes!  Autistic mice fed B. frag were less anxious, less obsessive, more communicative and interacted more with other mice.  The test for obsessive behavior was kind of cute.  The mice were put in a cage filled with sand with marbles sitting on top.  The autistic-like mice bury a greater percentage of the marbles, demonstrating a stereotyped behavior.

Yogurt from everyone!

If I had an autistic child and read this paper, I would start them on probiotics right away.  I mean probiotics are good for everyone, right, so it definitely seems worth trying.  In fact, the authors say that B. fragilis is depleted in human ASD children compared to matched controls.  Furthermore, probiotics have already been shown to be beneficial in treating chronic fatigue syndrome.   

The authors end their paper with this bold statement: “We propose the transformative concept that autism, and likely other behavioral conditions, are potentially diseases involving the gut that ultimately impact the immune, metabolic, and nervous systems, and that microbiome-mediated therapies may be a safe and effective treatment for these neurodevelopmental disorders.”

Throw another adipocyte on the fire

Humans are able to live in so many different climates, in a wide range of temperatures and yet our inner core body temperature remains nearly constant.  This ability to thermoregulate has something to do, of course, with clothing and the ability to cool and heat our living spaces, but our bodies also offer many adaptations to regulate body temperature.  If it’s too hot, we sweat, releasing excess heat through evaporative cooling.  If it’s too cold, we shiver, producing heat in our working muscles.  The production of heat through physiological mechanisms is called thermogenesis and also includes a non-shivering version.  Today’s paper is about non-shivering thermogenesis, which is when our fat cells produce heat.

Non-shivering thermogenesis

To understand how non-shivering thermogenesis works, we need to take a step back and discuss cellular respiration.  The cells of our body store energy from food in the chemical bonds of a molecule called ATP.  During cellular respiration, a cell will convert glucose or fat into carbon dioxide, while slowly tapping into the energy in those food molecules in order to make ATP.  The final step of cellular respiration is that the energy from the electrons in glucose are passed from protein to protein, releasing energy that is used to pump protons into a membrane-bound cellular space.  You can think of these protons as a form of potential energy, like stuffing a closet full of balls.  When you open up the closet door, all the balls come tumbling out, releasing their potential energy in the process.  During cellular respiration, this potential energy is used by an enzyme to make ATP.  During non-shivering thermogenesis, though, the potential energy stored in all those protons stuffed into a small space is released by the cell as heat.  Thus, the energy from food is used to heat the body rather than being stored in ATP.

The main type of cell that does non-shivering thermogenesis is brown adipocytes, or fat cells.  Brown fat is very common in infants, but is also found in adult humans in the upper chest and neck.  The purpose of brown fat is to provide heat for the body.  Thus, non-shivering thermogenesis is activated by a drop in body temperature.  The cold temperature is sensed by the brain, which activates the sympathetic nervous system (the “fight or flight” response), which signals to the brown fat cells to express the genes necessary to bypass ATP production and release heat instead.  In a recent paper published in PNAS, Ye et al. describe how a different type of fat cell is able to skip all the nervous system steps and sense the cold directly (red arrow in diagram).  It is pretty cool that the fat cells are able to sense temperature, as if they were neurons, and can act autonomously to remedy the situation.  No need for a brain here!

Independent thermogenesis

Through a series of experiments, the authors demonstrate that a particular type of fat cell will express genes necessary for non-shivering thermogenesis when exposed to cold, independent of sympathetic nervous system activation.

In one experiment, they grew fat cells at different temperatures and measured gene expression using a technique called quantitative PCR (qPCR).  The idea behind this technique is that if a gene is highly expressed, there will be a lot of mRNA in the cell (remember the “central dogma” of molecular biology) and qPCR is a method for measuring the concentration of mRNA for a particular gene.  They focused their measurements on thermogenic genes that are known to be part of the non-shivering thermogenesis mechanism, such as Ucp1, which is the enzyme that actually allows the protons to fall back across the membrane, thereby releasing their energy as heat.  They found that these fat cells that were exposed to the cold expressed more Ucp1 mRNA, even in the absence of any nervous system.  These are just cells in a dish, so this must be an intrinsic property of fat cells.

It wasn’t just any fat cell that had this response.  In fact, brown adipocytes did not express more Ucp1 in the cold.  It was a different type of fat cell called a white adipocyte.  What is white fat?  The majority of fat in our body is white fat and its purpose is to store fat for energy (for cellular respiration) and to act as a thermal insulator, so we don’t lose as much heat through our skin.  There is one subtype of white fat that has been shown to do non-shivering thermogenesis and it was this type that could express thermogenic genes, like Ucp1, in the cold, independent of the nervous system.

Okay, so these white fat cells don’t need input from the nervous system, but do they still use the same intracellular pathway to turn on expression of these genes?  Normally, when a fat cell is activated by the sympathetic nervous system, it sets off a molecular cascade of events inside the cell, which involves activation of molecules in a pathway called the cAMP pathway (as shown in the diagram).  The authors inhibited this pathway in various ways and found that the cells could still respond to the cold as before, so this effect must use a different pathway.

There are still a number of open questions, such as: how do fat cells sense temperature?  Do they use the same types of receptors as temperature-sensitive neurons?  Why are some white fat cells independent, but brown fat cells need the nervous system to activate thermogenesis?  One thing that is clear, however, is that white fat cells are clearly important for temperature regulation as well as fat storage.  The authors suggest that tapping into thermogenesis might be a good way to help obese patients get rid of excess energy storage by releasing it as heat.  This pathway that is independent of the sympathetic nervous system could allow medications to target only the fat cells without involving the sympathetic nervous system which controls so many other functions in the body.

Something to think about as the cold Bay Area summer sets in.