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!

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 

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.

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.

(Insert mildly provocative title here)

Ever seen a pair of pigeons going at it?  And did you notice a penis on the male pigeon?  The answer is no, because most birds do not have external genitalia large enough for penetration.  And yet birds reproduce via internal fertilization.  Why would evolution favor male genitalia too small to actually enter into the female?  This just seems so inefficient. 

There are a few birds that do have well developed phalluses, such as the duck and goose.  What happened during evolution that caused some birds to retain a phallus, whereas most other birds lost it?  A paper appeared this week in Current Biology by Herrera et al., which addresses these questions from a developmental point of view.

Developmental arrest

The authors started this study by comparing the development of the phallus in embryos of two different birds.  They chose to look at (1) chick embryos, which are part of the galliformes group of birds and have reduced phalluses and (2) duck embryos, which are part of the anseriforms group, which have well developed, penetrating penises.  They followed the growth of the genital tubercle, the tissue that will form the penis.  As the duck and chick embryos grow, so do their genital tubercles, with no noticeable difference between the two species during the early stages of development.  At a later time period, though, the tubercle stops growing and regresses in the chicks, while the duck keeps on growing.  This shows that the tissue that makes the two different types of phalluses has the same developmental origin.

Why does the genital tubercle stop growing in the chick?

From a molecular stand point, the chick embryos could either lose the “growth” signal or they could gain expression of a “stop” signal not present in ducks.  From work in other animals, the authors knew that there are two major growth signals responsible for guiding the development of the external genitalia – Sonic Hedgehog (Shh) [see my other post about this protein] and Hox13.  These two genes are strongly expressed in the duck genital tubercle throughout embryonic development, as expected.  Surprisingly, though, they are also strongly expressed in the chick embryos.  This means that the chickens haven’t lost the growth signal.

The authors then investigated if there is some sort of a “stop” signal in the chicks.  They found that in chicks and quails, with reduced phalluses, there is a lot of cell death in the genital tubercle in the later stages of development.  This could account for the regression of the genital tubercle.  They then found that the chicks highly express a protein called BMP4 at the tip of the tubercle, which induces cell death, whereas ducks do not. 

In fact, by overexpressing BMPs in the duck, they induced cell death in the genital tubercle.  In the opposite experiment, they inhibited BMPs in the chick and their genital tubercles increased growth, as if they were ducks.

In summary:

            Chicken: + BMP --> increased cell death --> reduced phallus

            Duck:       - BMP --> no cell death, so continued tissue development --> large phallus

Evolution of reduced phallus

So what does this mean?  Chicks and quails have reduced phalluses, because during development, they express BMP4, which tells the developing cells of the penis to die off.  One really cool thing that the authors did next was to look at cell death in the closest relative to birds-- the alligator.  Yah, they got alligator embryos for this research!  Alligators have developed phalluses and they show hardly any cell death in the genital tubercle.  From this work, they could create an evolutionary tree, which shows that chicks and quails most likely evolved the BMP4 signal after their group separated from ducks.  Although the authors didn’t test any members (ha ha) from the neoaves group, which includes most other birds, we can presume that they also have a similar cell death mechanism to reduce the development of their phalluses.

Phylogenetic tree of birds, showing when the BMP signal evolved. (Adapted from Herrera et al., 2013)

This still begs the question of why would natural selection favor a reduced phallus so much so that it evolved independently in different lineages?  The authors propose two different theories, both of which may have occurred:

1) Sexual selection – sure, it may not be favorable for the males to have reduced phalluses, but it might be advantageous for the females.  In order for insemination to occur in these species, the female has to be a willing participant to allow the male to shimmy up next to her and release the sperm in very close proximity.  This gives the females the power to select their mates.  As opposed to species with large penises, where the male could basically rape the female and still successfully pass on his genes to the next generation.

2) Pleiotropy – this term refers to when a single gene mutation can lead to multiple noticeable changes in the body.  BMPs are a major signal during development of animals.  BMPs are involved in a number of bird-only innovations such as feathers and beaks.  Maybe increased BMP expression gave an advantage to these birds, but also lead to reduced phalluses, as a secondary effect.  This may have occurred first in evolution, but sexual selection may have stabilized this characteristic in the population.

This article was so clear and interesting.  I’m sure it will catch people’s attention because of the subject matter, but it’s a great example of using development to solve an evolutionary question.  Plus it gives reviewers and bloggers a great opportunity to think up clever titles and puns for their articles.  The review that was published alongside this article was titled “Cock-a-doodle-don’t”. How can I compete with that?

Stop seizures with a brain graft

There are two types of neurons in the brain: excitatory and inhibitory neurons.  They do exactly what you think they would.  Excitatory neurons release chemical messengers, which activate other neurons, which may eventually lead to some sort of perception or action.  Inhibitory neurons release chemicals that silence other neurons.  Why would you want inhibitory neurons in your brain?  Well, if all your neurons were excitatory and interconnected, all your neurons would be active all the time and the signals would be meaningless.  In fact, this sort of overactivation in the brain can lead to seizures.  It’s been shown in numerous cases of epilepsy that there is some sort of dysfunction of the inhibitory neurons.  The excitatory neurons have free reign and go crazy, leading to a seizure.

How is epilepsy treated?  Medications that potentiate the inhibitory neurons can help, but they activate all inhibitory neurons throughout the brain, when maybe the problem is more localized to one spot.  Just as all excitatory neurons is a bad thing, too much inhibition is also bad and can lead to cognitive side effects.  Another treatment is to open up the patient’s head, try to find the overactive area and cut it out or zap those neurons with a laser.  Destroying brain cells is always a last resort, though.

In a recent paper published in Nature Neuroscience by Hunt et al., the authors propose another potential treatment: adding new inhibitory neurons into the epileptic brain.  Like all new medical ideas, the story starts with mice.  They can create a model of human epilepsy in these mice by treating them with a potent drug.  These epileptic mice have seizures just like humans do.

Where do you get new inhibitory neurons?

The researchers obtained progenitor cells from mice embryos.  In other words, these weren’t inhibitory neurons yet, but they were destined to turn into them as the mice developed.  They grafted these progenitors into adult epileptic mice in the hippocampal region of the brain (a common area for seizures).   Amazingly, these pre-neurons migrated throughout the brain region, as far as 1.5 mm (that’s a lot… think about how small a mouse brain is).  Then the progenitors differentiated into inhibitory neurons, as if they were in a normal developing brain.  One week later, the epileptic mice with extra inhibitory neurons had hardly any seizures, whereas the untreated mice were having about 2 a day.  Not only that, but the treated mice showed cognitive improvements compared to the untreated epileptic mice. 

So they seemed to “cure” the epileptic mice by giving them some new inhibitory neurons that were able to make functional connections with the existing neurons.  This isn’t as invasive as brain surgery and it’s much more localized than medication.  If the epilepsy were focused in a different part of the brain, then they could transplant the cells there instead.

Is this possible to try in humans?  Maybe so, but the first problem is that we can’t take inhibitory progenitor cells from human embryos.  There are some ethical issues with growing clones to harvest parts from them.  However, you could use embryonic stem cells, or induced pluripotent stem cells.  Pluri-what?  Recent technology allows researchers to take a skin biopsy, do some genetic engineering to these cells and push them back in developmental time to a stem cell.  Pluripotent means that these stem cells have the potential to become any type of cell, like an inhibitory neuron.  All it takes is turning on the right genes in these cells to push them to a particular fate, and if that isn’t already known for inhibitory neurons, I bet it’s not too far off.  Plus there’s the benefit that the transplanted cells will have the same genome as all the patient’s other cells, because they originated from their skin cells.  Just wait, regenerative medicine is moving ahead at lightning speed.

Go go gadget extendo filopodia

I’m back from an intense semester of learning and teaching Developmental Biology.  One theme that emerged from my studies was that the development of organisms is centered around gene expression and cell to cell signaling.  Often times, one cell will differentiate into its mature form, and then release a signaling protein that tells neighboring cells what to develop into.  For instance, the nervous system is induced by signals released from the embryonic backbone.  There are a number of common signals that are used over and over throughout development, like BMP, Wnt and Shh.

A recent paper by Sanders et al., published in Nature, looked at how distant cells can signal to each other via the Shh pathway.  Unfortunately for Developmental Biology teachers everywhere, Shh stands for Sonic Hedgehog.  Oftentimes, strange or humorous gene names like this can be blamed on the fruit fly researchers who first discovered the gene, but in this case everyone is to blame.  This gene was originally discovered by researchers studying fruit fly embryonic development; they named the gene hedgehog because the mutant embryos had lots of tiny bristles all over, kind of like a hedgehog.  The mammalian researchers took it to the next ridiculous level, by naming the mammalian version of this gene Sonic Hedgehog.  The Shh protein is a secreted signal that binds receptors on other cells, which activate gene expression in the receiving cell.  Shh signaling is important for specifying many different cell fates, such as the different neurons in the spinal cord, the cells that become the vertebrae, as well as the formation of the digits of the hand.

Although Shh is secreted from the cell, it has chemical modifications that make it stick to the plasma membrane that surrounds the cell that released Shh.  How then can Shh induce the development of cells that are located at a distance?  Well, the answer is by stretching out long cellular extensions with Shh localized at the tip.

Shh Filopodia

Sanders et al. did live imaging of cells in the developing limb of the chicken using fluorescent proteins.  They did some genetic trickery so only a few cells were labeled in red and others in green.  This way they could detect individual cells in a sea of unlabeled cells and examine their structure in real time.  They observed individual cells extending long protrusions, called filopodia, from the cell bodies.  These filopodia could stretch long distances (150 micrometers, like 3-5 cell widths) and were dynamic-- retracting and growing over time. 

How to think about filopodia?  Imagine a stretchy balloon with a stick inside of it.  If you could push that stick into the wall of the balloon, the balloon would protrude from that one spot as the stick pushes it out.  That is like a filopodia, where the balloon wall is the plasma membrane and the stick is a protein called Actin.  Actin forms long chains that can grow, pushing out the membrane in front of it. 

The thin, string-like extensions from this cell are filopodia and are filled with Actin.  Image from proteopedia.org

The authors then labeled the Shh protein with another fluorescent marker and saw that it localized to the tips of filopodia.  Not only that, but the filopodia expressing Shh were more stable and did not retract as often.  In order for Shh to act as a signaling molecule, it has to bind a receptor on another cell.  Using a different color, the authors observed two co-receptors for Shh localized to filopodia from other cells.  They even saw filopodia from two different cells make contact with each other, where one cell expressed Shh and the other expressed the receptors.

This is amazing!  Instead of releasing a signal out of the cell with the hope that it goes to the right place and isn’t degraded, the cells literally grow to the right place with the signal on their membranes.  This is like hand delivering a note to your coworker, rather than making the note into a paper airplane and throwing it in the direction of their desk.

This is how I imagine this working.  Two cells that are located at a distance, reach out extensions and meet somewhere in the middle. The Shh signal would bind the receptor, causing changes to the pink cell.

A study like this could not have been done before recent innovations in live imaging and molecular biology to introduce the fluorescent proteins into the cells.  The filopodia are not preserved during the more traditional, static method of fixing cells with formaldehyde and then staining them.  Who knows what other tricks live cells use during embryonic development.  I suspect this is only the beginning.

Swapping eggs

This week’s paper describes a new technique that could be used to manipulate human oocytes (i.e. eggs) to prevent a group of diseases called mitochondrial diseases.  The paper was presented by Tachibana et al. in Nature along with a similar paper by Paull et al.  For the sake of brevity, I will only discuss the findings from the first paper.

Mitochondria

So what are mitochondria?  Mitochondria are little compartments in the cell that make cellular energy.  They convert the energy stored in food into an energy source that the cell can use to drive chemical reactions.  In other words, they are absolutely essential for our survival.  The oxygen that we breathe in goes to the mitochondria to aid in this energy conversion, and we all know how vital oxygen is. 

There are two other interesting facts about mitochondria that relate to our story:

1) All the mitochondria in our body are duplicates of the mitochondria that were in our mother’s egg.  In other words, embryonic mitochondria are not made from our genomic DNA (gDNA) or from sperm contributions.

2) Mitochondria have their own DNA , which directs the synthesis of proteins that are necessary for their function.  This DNA is known as mitochondrial DNA (mtDNA) and it is only inherited from the mother, since all mitochondria originate from the egg.

If there are mutations in the mtDNA, then this can lead to problems with the synthesis of cellular energy, which can lead to human diseases known as mitochondrial diseases.  There are different types of mutations, which can affect people in different ways and with differing severities.  In this paper, the authors propose a way to prevent mitochondrial diseases from being inherited from generation to generation.  Let’s see how that works.

Nuclear transplantation

Let’s say you have a female patient with a mitochondrial disease, who wants to have a healthy child.  She is guaranteed to pass this disease on to her child via the mitochondria in her oocytes.  However, most of what makes the child “hers” is what lies in the mother’s genomic DNA, not in the mitochondrial DNA.  What if you could take the mother’s genomic DNA (plus the DNA from the father) and stick it into a healthy “enucleated” oocyte from a donor who has good, functioning mitochondria?  All the genomic DNA will have to be cleared out of the donated oocyte first, creating an enucleated egg.  The embryo that results from this nuclear transplantation will have genomic DNA from its mother and father, but its mitochondria will originate from the donor oocyte.  This would circumvent the mutated mtDNA that is in the real mother’s oocyte.

Tachibana et al. obtained human oocytes from volunteers and transfered the genomic DNA from one into another.  They then injected these oocytes with sperm DNA (like during real fertilization) and observed what happened.  Some oocytes failed to be fertilized and others died soon after, but a handful of oocytes survived into the blastula stage of development.  You can’t really grow a human embryo in a dish beyond the blastula stage and they are not allowed (yet) to implant these into women, so we don’t know what would happen to a child born from this procedure. 

They did carry out the above scenario with monkeys.  They transplanted the genomic DNA from one oocyte into another and implanted the blastula into another female monkey who carried the embryo to term.  The monkey youths are 3 years old now and doing just fine.  Their maternal genomic DNA is from one mother and their mitochondria are from a different oocyte donor. 

Isn’t this amazing?  I seriously doubt this procedure will be approved for human use anytime soon, because it’s too much like cloning, which basically follows the same procedure of putting genomic DNA into an enucleated egg.  It's a cool idea, though.