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.

Hot times are a-bloomin'

Remember a month ago, before the rains started, when it was warm and sunny and all the trees in Berkeley were blooming?  The Magnolias and plum trees were particularly beautiful. 

From Berkeleyside blog

How do the plants know that it’s warm and time to bloom?  Plants can sense day length and will begin to grow and bloom when the length of sunlight increases in spring.  Our trees were blooming in January and February, though, so plants must also be able to sense temperature changes too.  This week’s paper addresses the molecular changes that occur to induce flower growth in response to warmer temperatures, even when the daylight is still reduced in winter.  The paper by Kumar et al. was published online in Nature.

The transcription factor PIF4

The authors focused their studies on a protein called PIF4.  This protein binds to DNA and helps direct gene expression (or transcription), which makes it a transcription factor.  In normal, wildtype Arabidopsis plants, there is early flowering at warm temperatures, but in mutants that do not have PIF4, flowering is delayed.  In contrast, when PIF4 is overexpressed, early flowering is induced.  These results show that flowering at warm temperatures depends on PIF4.

The authors go on to show that PIF4 binds DNA just upstream of a gene called FT, which is necessary for flowering.  One can imagine a simple pathway where higher temperatures induce PIF4 to bind to the FT gene and activate gene expression.  Increased FT then initiates cellular pathways that signal the plant to go into bloom.

How do increased temperatures increase PIF4 binding to the FT gene?

1) Maybe high temperatures increase transcription of PIF4.  The authors measure RNA levels of PIF4 at different temperatures and find that there is more expression at higher temperatures, but not enough to account for the extreme differences in flowering.

2) Maybe high temperatures make the protein more stable.  In this case you would expect to see less PIF4 protein at cold temperatures, but the authors find that protein levels are unchanged by temperature.

3) Do high temperatures help the binding interaction between PIF4 and FT?  Yes, there is more PIF4 bound to FT at higher temperatures, but why?

Nucleosome H2A.Z

In past posts, I’ve talked about histones in the context of epigenetics.  DNA wraps around histone proteins to help pack the DNA into a smaller package.  The collection of histones that DNA wraps around is called a nucleosome.  When DNA is wrapped around nucleosomes, other proteins cannot bind to the DNA and gene expression doesn’t happen.  There is a nucleosome called H2A.Z that binds near the FT gene in the same place where PIF4 needs to bind.  Therefore, when the nucleosome is present, PIF4 cannot bind and activate expression of FT and there will be no flowering.  H2A.Z binding to FT is temperature dependent in the exact way you would expect.  In cool temperatures, H2A.Z binds FT DNA, so PIF4 cannot bind and FT will not be expressed.  In warm temperatures, for whatever reason, the H2A.Z nucleosome comes off of the DNA, allowing PIF4 to bind.

 

Adapted from Figure 4C of Kumar et al., 2012, Nature.

It’s nice when there is a relatively simple molecular explanation for something we observe in the environment all the time.  This subject is also important because global warming will continue to induce earlier and earlier flower blooming, which could be detrimental to food crops.