Wednesday, January 15, 2014

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

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 (Xt) and one X with GFP (XG).  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.