Monday, January 5, 2015

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 12th 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.

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