Hi, I'm Abby Dernburg.
I'm on the faculty at UC Berkeley,
and today I'm gonna give you an introduction to meiosis,
the specialized cell division
that gives rise to sex cells
like sperm, or eggs, or pollen.
People started noticing, in the mid 19th century,
when they started looking through the microscope at stained cells,
they would notice these dark bodies
in the nucleus.
And they started looking the process of cell division,
first looking at this process of mitosis,
that's much more familiar to most biologists
than meiosis.
During mitosis,
the goal is to replicate chromosomes
and then segregate them to daughter cells
so that you create two daughter cells
that are basically the same as the mother cell.
So, in order to accomplish this,
chromosomes undergo DNA replication
and then they condense so that we can actually see them,
they line up at the center of the cell,
this mitotic spindle forms,
and eventually the glue, the cohesion
that holds chromosomes together,
gets severed and allows the chromosomes to segregate
to opposite poles to create two new daughter cells.
So, meiosis is a little bit different.
So the fundamental goal of meiosis
is to take a cell that has two sets of chromosomes
-- the set that was inherited from the father
and the set from the mother --
and to divide those chromosomes
to produce cells that have only a single set of chromosomes,
and those haploid cells then go on
to develop as sperm, or eggs, or pollen, or spores.
So how does this specialized reductional cell division process work?
What really differentiates mitosis and meiosis
is the fact that the chromosomes that are gonna segregate
in the first meiotic division do not start out together.
So, in mitosis,
chromosomes undergo replication,
and so you have two sisters,
but they're held together by cohesion,
and they stay together until they're gonna segregate.
So they don't have to find each other.
But in meiosis, the goal, as I said,
is to separate the two copies of the chromosome
that you got from your two parents.
And in order for that to happen,
those chromosomes, which are called homologues,
have to first pair with each other.
And we don't know exactly how they do that,
I'll have more to say about that in other segments,
but somehow, through a mysterious process,
they come together, they find each other, they recognize each other,
and they pair up along their entire lengths,
and they form this mysterious sort of glue
that we call the synaptonemal complex
that holds them together for much of meiosis.
They also have to undergo recombination,
and that means that the DNA has to get cut and repaired
in a special way
that connects homologous chromosomes.
So, DNA is cut and it's repaired in a way
that physically connects homologous chromosomes.
And this process of recombination
is thought to be sort of the raison d'etre of meiosis;
it's thought to be the reason that eukaryotes
have taken over the earth
and have achieved such diversity and complexity of their form,
because they undergo recombination.
And the idea is that that enables mutations to arise
that are beneficial,
and for those mutations to be separated from the deleterious mutations,
which are much more common,
in an efficient way,
so that evolution can happen at an accelerated pace
compared to asexual reproduction.
And because this recombination process
is so central to what meiosis is all about,
it is also physically essential for this process.
Recombination is actually necessary
to create the link between chromosomes
that enables them to stay together
until they go and get ready to divide.
So, basically, by forming this crossover
and being held together by cohesion,
the two homologous chromosomes
can then bi-orient,
meaning they can face the two poles of the meiotic spindle,
and segregate towards opposite poles.
This whole process of pairing and synapsis and recombination
takes place during a long period
that we call meiotic prophase.
It can last anywhere from about 24 hours to many days,
depending on the organism and the complexity of the genome.
Once the chromosomes have accomplished
pairing, synapsis, and recombination,
they eventually get ready to divide,
they condense,
and they segregate in two successive cell divisions.
First, the homologous chromosomes separate from each other,
and then the sister chromatids come apart,
as they do during mitosis.
And this requires a lot of interesting variations
on what happens during mitosis.
In particular, in mitosis,
the sister kinetochores, the centromeres have to separate.
But here, they actually have to stay together
during the first division
and separate at the second division.
And likewise, during mitosis,
when chromosomes are ready to divide,
they simply release the cohesion that's holding them together,
but in meiosis that cohesion has to be released in two steps.
First partially, to allow the homologues to separate,
and then the rest of the cohesion
is released to allow the sisters to separate.
So all of this requires
modification of the mitotic cell cycle,
some of which we understand,
and some of which is still areas of very active research.
So, we're interested in meiosis
from the standpoint of understanding this very fundamental cellular process
that's shared by almost all eukaryotes,
and we're also interested in understanding
how errors in meiosis arise,
partly because they give rise to
many human birth defects.
And this is the best known example,
Down Syndrome, which is when
an infant inherits an extra copy
of a small chromosome, chromosome 21.
This almost always happens due to errors
in female meiosis, in humans.
Male meiosis, even though it happens much more abundantly,
humans make many more sperm than they make oocytes,
is actually much more faithful
and less error-prone
for reasons that we don't really understand.
So, we want to understand
how these chromosome segregation events occur,
what regulates the whole process,
and it's very difficult to study in humans,
partly because all of these meiotic events
-- pairing and recombination --
all happen in the ovaries
when a fetus is still in utero,
so that makes it kind of inaccessible.
On the other hand, it's quite easy to study in some model organisms,
and the one that my lab uses to study meiosis
is the nematode Caenorhabditis elegans.
It's a great experimental system.
These are small roundworms, they're very widely used in studies of
neural development and other developmental processes.
So, one of the great advantages of using C. elegans
to study meiosis has to do with just the anatomy of this animal.
There are very small animals, they're about a millimeter long.
In a wild population, most of the animals are hermaphrodites,
so they make both eggs and sperm,
and they fertilize their own eggs with their own sperm.
The animals are very small, they're about a millimeter long,
and most of the interior of an adult animal
is actually the germline,
the tissue that gives rise to meiotic nuclei, and eventually to the progeny.
The germline in this animal is organized within these two arms of the gonad,
that have a sort of horseshoe-like structure,
and what's wonderful about this system is that
the gonad contains a complete
gradient of meiotic stages,
and so it's very clear where each thing is happening within this animal.
In the distal region of the gonad,
there are these proliferative zones,
where nuclei are just dividing mitotically,
and then as nuclei sort of get pushed away
from the distal tip of the gonad
and move towards the uterus,
the nuclei enter meiosis,
they go through this stage that we call the transition zone,
which is where pairing and synapsis occur,
and they initiate meiotic recombination,
which is complete by mid-pachytene.
And then eventually the chromosomes
start to condense and they segregate.
First, they give rise to a pool of sperm,
and then oocytes which pass the through the spermatheca
get fertilized and the resulting cells
start dividing internally as embryos.
So it's very easy to see
where everything is happening during meiosis.
We can dissect out the gonads from adult animals
and stain them with a variety of reagents
to visualize specific proteins or DNA sequences
and, as I'll talk about in another segment,
we can also take advantage of the fact that
these animals are transparent,
and we can put them under a coverslip
and actually watch meiosis in living animals.
So, if we dissect out the gonad and stain it with, here,
a dye that binds to DNA called DAPI,
what we see is all the individual nuclei.
So that's what you're seeing here, each little sphere is a single nucleus.
What you're looking at... about...
you're looking at a projection through about half of this gonad.
It's actually a tube,
kind of like an ear of corn,
where the nuclei are arranged around sort of a central matrix.
And as I described,
here in the distal region there's this pool of
proliferating premeiotic nuclei,
and then we can sort of tell right when meiosis starts
'cause the nuclei take on a sort of different appearance,
they look more crescent-shaped if we look at the DNA.
We call that the transition zone,
and we know that's where pairing and synapsis occurs,
and recombination is initiated.
Recombination is completed, and then eventually, as I said,
the chromosomes condense, and at this point, in diakinesis,
what we see are these six little cruciform structures,
which are the six pairs of chromosomes
that are held together by the fact
that they're undergone crossover recombination.
So we can see everything that's happening,
and one way that we identify
the specific molecular components
that are involved in driving this process is through genetics.
And that turns out to be relatively easy in C. elegans,
partly because of the mechanism of sex determination
in this organism.
So, as I described,
in a wild population of C. elegans,
most animals are hermaphrodites,
and they have six pairs of chromosomes,
which is a really nice small number.
Humans, for example, have twenty-three pairs of chromosomes.
That's a lot more to contend with,
and so with six pairs of chromosomes,
it's quite easy to visualize each chromosome individually,
in the nucleus.
So, males are produced by C. elegans,
and that's very important for doing genetics,
because that's the only way we can cross two genotypes together.
They're usually at a low fraction of the population,
and males are basically the same as hermaphrodites,
except they have a single X chromosome.
So where do males come from?
So it turns out that in this organism
males arise through what are basically errors in meiosis.
And what I mean by that is if you have a hermaphrodite,
which has two X chromosomes,
if it goes through meiosis to make either sperm or oocytes,
what should happen is that that two X chromosomes should pair,
and they should segregate away from each other,
and every sperm and every oocyte
should get a single X chromosome.
And so when a sperm fertilizes an oocyte,
you should restore the XX chromosome content
that gives a hermaphrodite.
But it turns out that, at a low rate in a normal animal,
but at an elevated rate in meiotic mutants,
you can see missegregation of the X chromosome,
so the X chromosome will get lost or missegregated during meiosis,
and as a result you'll have a sperm or an oocyte
that doesn't have an X chromosome,
and at fertilization that can give rise to an XO,
or male animal.
So, in a normal animal that happens only
about 1 in 500 meioses give rise to
an oocyte or spermatocyte lacking an X chromosome.
In a meiotic mutant though,
what you get are far fewer viable progeny,
lots of males,
and lots of dead embryos that arise
because the other chromosomes are also missegregating,
and if an animal inherits the wrong number
of those chromosomes it's usually inviable.
So, many screens have given us a wealth of mutants,
they often are called 'Him' for High incidence of males mutants,
and they're often identified by putting individual worms on plates
and looking at their broods, their progeny,
to find ones that are throwing lots of males.
There's sort a cute shortcut that was invented by Anne Villeneuve,
which uses a clever way to identify animals
that are going to throw lots of males
without having to put them on individual plates.
It takes advantage of this xol-1::gfp reporter.
So xol-1 is a gene that's normally expressed
only in male embryos,
and if a hermaphrodite, which is what we're looking here,
is a Him mutant, so it's going to produce lots of males,
and it's got this xol-1::gfp reporter,
it's embryos, inside it,
will be green and fluorescing,
and so we can use that as a way to identify hermaphrodites
that are meiotic mutants,
and screen more efficiently.
And, as I said, Anne Villeneuve invented the screen
and it's called, after the Dr. Seuss book,
the "Green Eggs and Him" screen.
So that has given us lots of specific mutants
that have told us a lot about this meiotic process
and how it works.
So, one of the key things that my lab is interested in,
and many people in the field are interested in,
is this process of homologue pairing.
It's unique to meiosis,
how do chromosomes
find their partners inside the nucleus,
and what is it that tells them that
this is your partner and you should pair up with it?
I'm not gonna be able to answer those questions for you,
'cause we are still actively working on understanding this,
but I'll tell you a little bit about what we've learned.
And I'll start with something
we learned about how they don't pair.
So, back when I started working on meiosis,
we used to think of the process of meiotic prophase
in this very linear way.
We imagined that chromosomes, as I described,
would undergoing pairing and synapsis,
and then they would undergo recombination.
And the logic of that was obvious:
if you're sitting right next to your homologous chromosome,
then when your DNA is cut and you have to repair it,
you have a homologous chromosome right next to you
that you can undergo recombination with.
And so this seemed to make sense
as a way to kind of structure this process.
However, in the early 1990s,
Scott Keeney and Nancy Kleckner provided evidence
that this sort of linear view of this process
was probably not quite right,
at least in the budding yeast S. cerevisiae.
What they found is they identified
the enzyme that actually cuts the DNA
to initiate meiotic recombination,
it's called Spo11,
but what observed is that mutations in Spo11
don't just disrupt this process of recombination.
Instead, they actually disrupt the process of pairing and synapsis.
So that tells us that, in this budding yeast,
and it turns out also in plants and in mammals,
the process of pairing and synapsis
requires the early stages of meiotic recombination.
So when I started working on meiosis in C. elegans,
we didn't really know if this was the case,
and we wanted to test it.
And as I said, we have the advantage of
being able to actually observe the formation of recombination products,
these conjoined bivalent chromosomes,
and we were able to identify the Spo11 homologue,
the gene encoding the worm Spo11,
in the genome and to obtain a mutation in that gene.
And what we could see immediately was,
whereas in a wild type animal, as I described,
there are these 6...
each oocyte contains these 6...
these cruciform bivalent structures
which are the 6 chromosomes
that have undergone recombination.
When we looked at Spo11 mutants,
what we see is different.
In each oocyte, we see 12 univalent chromosomes,
meaning that they are not attached to their partner.
And that tells us that, as in budding yeast,
Spo11 is required to make crossovers.
We wanted to know if Spo11
was also required for the process of pairing and synapsis,
and we can monitor that in a variety of ways.
Here's one way that we do that.
This is just a blowup of a region of the gonad,
the region of the gonad where pairing and synapsis occurs,
I described it's called the transition zone,
and here we're looking at these orange spots,
which are due to in situ hybridization.
So we've made a probe that hybridizes with
a particular locus in the genome and,
if the chromosomes have not yet paired,
you see two spots.
But the time they're in pachytene,
down here at the right bottom,
they have all paired and synapsed,
and so every nucleus has just one big spot,
or a very close pair of spots.
And in this transition zone, that's where pairing and synapsis occur.
So it was very straight-forward for us to look at this Spo11 mutant,
and we could tell right away that
the chromosomes had no difficulty pairing and synapsing
in the absence of Spo11,
suggesting that things were a little bit different
than they are in budding yeast.
We needed to prove, though,
that Spo11 really just is only required
to make double-strand breaks in C. elegans,
and the way we tested that was basically the idea...
we tested that using an experiment
that recapitulated an old experiment in budding yeast,
in which we took Spo11 mutants
and we subjected them to radiation,
which of course breaks DNA.
And so the idea is that if Spo11 is only needed
to make double-strand breaks,
to make breaks during meiosis,
then radiation should partially substitute for that function.
And we were able to show that,
in Spo11 animals, if we irradiated them,
there was a burst of greater progeny viability
than in unirradiated animal.
And so, indeed, we could demonstrate
that the function of Spo11
is specifically to make double-strand breaks but, as I said,
it's dispensable for pairing and synapsis.
And this just shows, cytologically,
that when we irradiated Spo11 animals,
we were able to restore crossing-over,
and we could detect that as these bivalents in the oocytes.
Okay, so apparently pairing and synapsis in C. elegans
does not require double-strand break formation,
implying it really doesn't require
the recombination machinery
which acts at those double-strand break sites.
So, how do chromosomes pair and synapse?
And as I said, this is still a big mystery,
but I'll tell you in my additional segments
what we've learned about this process so far.