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What we talked about last time was
formation of different types of

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cells in the embryo,
different positions, different

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shapes.  Am I on?
And we talked about one of the

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overriding principles of all of this
being the control of gene expression.

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I'll explore this a little more
with a couple of slides in a moment.

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Let me just write some points.  So
we talked about the control of gene

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expression as being an overriding
principle of how cells

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types are formed.
We talked about the fact that cell

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types were formed in a stepwise
fashion.  And different parts of the

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embryo indeed in a stepwise fashion.
Not all at once.  And we talked

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about a combinatorial code --

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-- of regulators that,
together, specify different types of

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cells.
So here we are to remind you talking

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about cell type in the formation
module stepping through our board of

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life.  And here are a couple of
slides to remind you what we talked

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about in last lecture to bring you
up to speed.  You should look at the

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PowerPoint on the Web if you have
not already.  Cell type is defined

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by the proteins a particular
cell makes.

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For example, red blood cells carry
oxygen around the body because they

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make globin which is able to carry
oxygen.  The set of proteins that

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are made are defined by which genes
in a cell are active,

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or expressed is the term you should
know.  And, therefore,

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which genes are active,
also called the control of gene

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expression, controls which
cell type forms.

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This triad is essential for you to
understand what we're going to be

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talking about now and really for the
rest of the course.

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So you should really get it.
If you don't see me, go back and

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look at the previous lecture.
And this lecture is going to also

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reinforce this triad.
I pointed out last time that gene

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expression can be controlled
at many levels.

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In fact, at any point from
transcription,

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even from replication actually of
the DNA through transcription,

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splicing, translation, export of the
RNA to the cytoplasm,

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I haven't written up here,
messenger RNA stability, protein

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modification, protein export.
Any of these steps is along the way

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to getting the final gene product.
And any of these steps can be

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controlled to allow the readout of
the final gene product or not.

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I told you that along the way to
becoming the final type of cell that

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a cell is going to become,
it goes through a couple of steps.

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Or it can go through many steps,
and we'll talk about that

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extensively today.
Cells begin as uncommitted or naive.

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They don't know what they're going
to become.  They then become

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committed or determined to a
particular fate,

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a position or a structure,
but they may not look very different

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from that first set of
uncommitted cells.

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And then they go on eventually to
form so-called differentiated cells

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or the final cell type which has the
function of that particular cell

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type.  And so the point is if there
is a stepwise formation of the final

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cell type.  And that final cell type
is directed through the action of

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regulatory factors that we termed
inducers or determinants.

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There is nothing magic about these.
Inducers referred to factors that

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were involved in cell-cell signaling.
Determinants referred to regulatory

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factors that acted within a cell and
were inherited by it.

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You shouldn't be trying to write
all this down now actually because

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this is really review.
And if you weren't at the last

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lecture, try to get some of it to
wash over you and then go back and

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make sure you really
know this stuff.

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And then as the differentiated cells
form there is activation of a set of

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genes called differentiation genes
which are the things that carry out

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the final cell function,
the globin protein in red blood

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cells, the neurofilaments and
neurotransmitters in neurons and so

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on.  And we'll talk about this again
as we go through today.

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OK.  So this is not unrelated to
what we are going to

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talk about today.
Because what I want to do today is

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to explore with you how you get a
particular kind of cell.

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And I want to use that exploration
to underline some of the principles

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that I haven't yet covered as to how
cell type is determined in the

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embryo.  And the cell type I'm going
to talk to you about is skeletal

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muscle.  No, this is not
the [UNINTELLIGIBLE].

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OK.  So the point today is to look

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at a particular cell type and how it
forms.  The type we've chosen is

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skeletal muscle.
Skeletal muscle is one of three

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kinds of muscle in your body.
The other two being cardiac in your

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heart and smooth muscle around your
internal organs.

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Skeletal muscle acts in a voluntary
fashion to allow movement.

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It's an extremely complex structure.
The final form of skeletal muscle

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includes 200 proteins which have the
extraordinary property of

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contractility in response
to nerve input.

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I'm not going to talk about the
contractile process,

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although it's fascinating.
And you're not going to be tested

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on this for the physiology of
contractility either.

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But if you're interested in this,
I would suggest you look at Chapter

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47 in your book which talks about
this quite nicely.

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Skeletal muscles are arranged in
large groups of fibers.

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And each muscle fiber is a
multinucleate cell,

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is a giant cell that consists of
many myofibrils or myofibrils that

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are together in a sheath and are
surrounded by many nuclei.

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So skeletal muscle forms from the
fusion of many mononucleate cells to

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give a multinucleate syncytium.
And during the process of skeletal

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muscle formation there are a number
of steps that one can delineate.

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One can talk about a kind of cell
called a myoblast which is a single

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mononucleate cell --

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-- that then fuses to form,
through many steps, something called

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a myotube which is multinucleate.
And these various myotubes come

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together to eventually
form a muscle fiber.

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Muscle is obviously an extremely

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important cell type for movement.
All of the meat that you eat, if

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you're a meat eater,
is muscle, essentially a skeletal

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muscle.  And there are many diseases
that are associated with abnormal

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skeletal muscle.
Muscular dystrophy,

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for example, is a deficit of one
particular protein in the muscle,

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affects one in about 3,000 boys.  OK.
So the myofibril is a very

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beautiful construction of many
proteins organized in a very ordered

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structure.  And the deal is that
these proteins slide relative to one

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another during contractility.
And all of these bands of proteins

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slide in an ordered fashion relative
to one another as the muscle

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contracts.  It's an extraordinary
story in physiology itself.

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And I regret I don't have time to
go through it with you.

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But I want to use this rather as an
example of a cell type and to

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explore with you how the cell type
forms during development.

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So the story I'm going to tell you
has to do with a particular

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transcription factor called MyoD.
MyoD, shown in green here, no, not

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shown in green here.
That's the DNA double helix.

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Shown in red here binds to a
particular sequence on

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DNA, its target site.
It binds as a dimer.

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And it acts to activate the
transcription of genes required for

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muscle formation.
OK.  So MyoD is a transcription

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factor.  And, as I'll tell you a
moment, it gives rise to the

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determination of skeletal muscle.
Now, I'm going to tell you a few

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things about this that are general
principles of how this transcription

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factor works and then I'll go
through some slides with you.

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MyoD is both necessary, although
I'm going to clarify that in a

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moment, and sufficient for
muscle determination.

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This is a dyad of words that is very
important, necessary: required for,

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sufficient: enough to.  And whenever
you're thinking about gene function,

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this is the dyad you want to think
about.  It may not be a gene,

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and then I'll tell you in a moment.
I'm going to put a caveat here with

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respect to the necessary,
OK?  But necessary and sufficient is

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one of these litanies that you want
to ask yourself when you're thinking

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about regulatory factors.
The other thing that I want to tell

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you is that MyoD is a member of a
gene family.  What's a gene family?

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A gene family is a group of genes
with similar structure and often

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similar function.
This gene family that MyoD belongs

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to is called the MRF gene family,
which stands for muscle regulatory

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family.  And there are
four members.

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And these members share some

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function with one another.
And that leads us to another term

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which is the term redundant.
MyoD is an extremely important gene.

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And if you make knockouts in mice
there is some phenotype,

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but they have muscle.
And it's not until you make a gene

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mutation in another member of the
MRF family that you see mice that

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don't have any skeletal muscle.
So redundant in this case refers to,

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or in all cases refers to shared
function.  It can also refer to,

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and does in this case, it can also
refer to alternate pathways.

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So the notion that we have is that
we're starting here as an

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uncommitted cell and we're going to
end up over there as a

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differentiated muscle cell.
And we can get there by walking

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straight this way,
but we could also get there by

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walking up and out the back and all
the way around and to that same spot

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in front.  And both of those would
be bona fide pathways that would get

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you from point A to point B.
So sometimes there can be alternate

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pathways that will get you along the
path of forming particular cell

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types or even carrying out some kind
of biochemical function.

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OK?  This is a general principle of
biology.  So let's look at this in a

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bit more detail with some slides.
The notion is that MyoD binds to the

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promoters of many different genes
expressed in skeletal muscle,

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together with other transcription
factors, some of which may be common

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to all of these genes and some of
which may be different.

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And it activates their
transcription.

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And you can see I haven't drawn the
genes in a really accurate way.

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These are just representations.
There's no double-stranded DNA

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meant to be implied or
not implied there.

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MyoD is sufficient to activate the
skeletal muscle program.

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And this is a very exciting finding
about 15 years ago when it was shown

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that one could take the MyoD gene,
and you actually put it in a virus,

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and then you can infect a group of
cells that normally wouldn't become

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muscle with this virus.
And lo and behold the virus that is

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expressing the MyoD protein will
turn these cells into

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skeletal muscle.
And this was the first example of a

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regulatory protein that could take
one cell type and turn it into

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another cell type.
And MyoD and the other MRFs are

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very good at doing this.
They can, for example, take brain

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cells and turn those into muscle.
They can really take most cell

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types and turn them into muscle
cells.  So MyoD is sufficient to

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convert non-muscle to muscle cells.
And this is actually particularly

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extraordinary because,
as I showed you in a one line

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schematic on this board,
forming skeletal muscle is a

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multi-step program.
You start off with these cells

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called, they call them myotome cells,
and then you get these things called

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myoblasts which are dividing.
The myoblasts start to align in

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arrays.  And then the cells fuse
with one another,

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which is really unusual for cells.
I mean it's a really kind of weird

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cell type.  And you get these long
tubes that have got thousands of

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nuclei in them.
And then the tubes start to make

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proteins that align themselves in
these very ordered ways that allow

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them to slide relative to one
another, and you get this

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multinucleate myotube that has
contractile function.

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And what's really cool about MyoD is
that it can start this whole process

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which then follows one step after
another.  So it's been termed a

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master regulatory switch because it
can activate a whole cell

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type specific program.

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Now, in contrast,
and this is what I was alluding to

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on the board, knockout or removal,
a genetic mutation of MyoD does not

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remove skeletal muscle from mice.
So this is a micrograph of muscle.

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You can see the ordered myotubes
from a wild type mouse.

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This is a MyoD null mouse.
And there's perfectly fine skeletal

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muscle there.  It's a little,
there's a little less of it, but

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it's still there and the mouse is
viable.  However,

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if you go in and you make a double
mutation in the MyoD gene and

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another member of the family called
Mif5, there is no muscle at all in

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any region, or no skeletal muscle at
all in any region of the body.

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The other muscle,
the heart muscle and the muscle

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around the intestine,
the smooth muscle is fine,

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but the skeletal muscle is
completely absent.

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So we can say that the MRFs are
necessary for muscle formation but

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that MyoD is a member of a redundant
gene family.  And that seems to be

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the rule in biology,
that genes come in families,

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and very often one can take over the
function of another when one of them

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is mutated.  And that serves as a
kind of fail-safe for making sure

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that development works properly.

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OK.  So here comes something to
think about that I alluded to before

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spring break.  I remember this.
Don't know if you do.  MyoD is,

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in the entire embryo the MyoD gene
is only expressed in the future

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skeletal muscle.
This is a picture of a frog embryo.

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And you can see these stripes along
the back of the frog.

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These are regions of the embryo
called the myotones.

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They're part of the somites that
I'll talk about.

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And this is what's going to become
the future skeletal muscle.

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And this is where MyoD is expressed.
And, brilliant,

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only there.  And now this is a
chicken embryo and you see the same

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thing.  MyoD is expressed in these
stripes along the back in the future

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00:19:27,000 --> 00:19:31,000
myotones of the somites.
It's also expressed in the future

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00:19:31,000 --> 00:19:35,000
limbs as the muscle of the limbs is
starting to form.

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And so you're going to ask me,
or you should, how does MyoD get

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activated just in the future
skeletal muscle?

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Because in a way this is a copout.
I've told you MyoD activates the

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skeletal muscle program,
and it's only expressed in cells

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that are future skeletal muscle.
Well, how does it know to be

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expressed just in this one
region of the embryo?

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00:20:00,000 --> 00:20:05,000
And to answer that question we have
to actually go back to the dawn of

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00:20:05,000 --> 00:20:10,000
time to fertilization.
And I'm going to take you through

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some of the steps that will get MyoD
expression into this particular

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pattern that will allow the skeletal
muscle to grow out in just the right

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places of the embryo to give just
the right amount and the right

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position of skeletal muscle.
OK.  So.  And to do that we are

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going to pose some questions.
And I'm going to start by telling

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you something and then pose a
question.  If you look at an embryo,

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you could look back at that chick
one if you wanted to do or you can

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00:20:48,000 --> 00:20:53,000
look at this fish.
Here is your fish fillet,

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00:20:53,000 --> 00:20:58,000
your skeletal muscle, and it is on
the so-called dorsal or backside

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00:20:58,000 --> 00:21:03,000
of the embryo.
So last time we talked about dorsal

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and ventral as two of the so-called
axis or positional coordinates in

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00:21:08,000 --> 00:21:13,000
the embryo.  And the muscle arises
from a region that is termed dorsal

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00:21:13,000 --> 00:21:18,000
which is the back.
OK?  So you might ask me,

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00:21:18,000 --> 00:21:23,000
how do you know this?  And I'll tell
you how I know this.

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You should ask me how do I know
this.  OK, so I'm going to.

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Having put questions into your
mouths, I'm going to tell you how

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00:21:33,000 --> 00:21:39,000
you know during the development of
an organism where a particular cell

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00:21:39,000 --> 00:21:45,000
type is going to arise from.
And the technique I'm going to tell

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00:21:45,000 --> 00:21:51,000
you about is something called fate
mapping.  So let's begin with the

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00:21:51,000 --> 00:21:57,000
sentence muscle,
and I'm talking about skeletal

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00:21:57,000 --> 00:22:03,000
muscle when I write muscle,
arises from the dorsal or the back

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00:22:03,000 --> 00:22:08,000
of the embryo.
OK?  And so how do you know this?

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00:22:08,000 --> 00:22:14,000
How is this known?  And it's known
through use of a technique called

255
00:22:14,000 --> 00:22:20,000
fate mapping.  What is fate mapping?
So this is very interesting.  The

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00:22:20,000 --> 00:22:25,000
idea is to take a very early embryo,
I've shown you here a fish embryo,

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00:22:25,000 --> 00:22:31,000
and to inject one or a few of its
cells that you can distinguish by

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00:22:31,000 --> 00:22:37,000
some kind of morphological,
obvious indicator from the rest of

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00:22:37,000 --> 00:22:42,000
the embryo.
And to inject those cells with a dye.

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00:22:42,000 --> 00:22:47,000
So here we've used a fluorescent
dye.  And we've put that dye near

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the site of the embryo that's got
this bump.  OK?

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00:22:52,000 --> 00:22:57,000
And as the embryo develops the
cells will inherit that dye because

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00:22:57,000 --> 00:23:02,000
it's a non-diffusible dye.
And that dye will be inherited.

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00:23:02,000 --> 00:23:07,000
And in the later fish, we're
looking down now onto its brain,

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00:23:07,000 --> 00:23:12,000
you can see that the whole front of
the brain is outlined with these

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green fluorescent cells.
And what this tells you is that the

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00:23:16,000 --> 00:23:21,000
cell you injected initially has gone
on to give rise to this whole front

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00:23:21,000 --> 00:23:26,000
region of the brain.
OK?  So fate mapping tells you what

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00:23:26,000 --> 00:23:37,000
cells will become.

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00:23:37,000 --> 00:23:41,000
All right.  I know it's on the slide,
but it's important that you

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00:23:41,000 --> 00:23:45,000
understand this because it is
distinguished from something I'm

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00:23:45,000 --> 00:23:49,000
going to tell you later.
So you can do this for skeletal

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00:23:49,000 --> 00:23:53,000
muscle.  And I'm going to talk about
frog development because so much is

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00:23:53,000 --> 00:23:57,000
known.  So here is a frog at the 32
cell stage.  And one can see,

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because of the size of the cells and
because of pigmentation differences

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00:24:01,000 --> 00:24:05,000
I'll show you in a moment,
which side of the embryo is what.

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00:24:05,000 --> 00:24:09,000
And I've injected a cell here that's
called the C3 cell,

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00:24:09,000 --> 00:24:13,000
all these cells have got different
names, with dye.

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00:24:13,000 --> 00:24:17,000
And later on you can see,
in this thought experiment,

280
00:24:17,000 --> 00:24:21,000
that the somites are labeled with
the blue dye.  So the C3 cell,

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00:24:21,000 --> 00:24:25,000
and actually the one on the other
side of embryo,

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00:24:25,000 --> 00:24:30,000
are going to give rise to skeletal
muscle.  OK.

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00:24:30,000 --> 00:24:34,000
So the reason that I wrote on the
board what was on the slide already

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00:24:34,000 --> 00:24:39,000
is that it's really important to
distinguish what cells are going to

285
00:24:39,000 --> 00:24:43,000
become from when they've made the
decision to become it.

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00:24:43,000 --> 00:24:48,000
So the next thing we want to know
is when cells decide what they're

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00:24:48,000 --> 00:24:52,000
going to become.
And I can rephrase that by saying

288
00:24:52,000 --> 00:25:01,000
when is the fate decision made?

289
00:25:01,000 --> 00:25:06,000
And fate mapping doesn't tell you
that.  All it tells you is what a

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00:25:06,000 --> 00:25:12,000
cell is going to become,
not when the cell has decided to

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00:25:12,000 --> 00:25:18,000
become that thing.
For this you have to do some other

292
00:25:18,000 --> 00:25:24,000
assay, and you do assays that are
generally called determination

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00:25:24,000 --> 00:25:30,000
assays.  And I'll show you an
example of one such assay.

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00:25:30,000 --> 00:25:34,000
So here's our 32 cell stage frog
embryo again with the C3 cell

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00:25:34,000 --> 00:25:38,000
labeled.  And one can go into the
embryo and dissect out that cell.

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00:25:38,000 --> 00:25:42,000
Frog embryos are about a millimeter
in diameter.  And you can use very

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00:25:42,000 --> 00:25:47,000
fine glass needles to tease out that
cell from the embryo.

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00:25:47,000 --> 00:25:51,000
And you can grow it in a very
simple culture method with just some

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00:25:51,000 --> 00:25:55,000
saline solution and the cell will
grow fine and divide.

300
00:25:55,000 --> 00:26:00,000
And you can ask whether that cell
goes on to make skeletal muscle.

301
00:26:00,000 --> 00:26:04,000
OK?  Does it know that it's going to
be skeletal muscle?

302
00:26:04,000 --> 00:26:08,000
And the answer at this stage of
development is no,

303
00:26:08,000 --> 00:26:13,000
it doesn't.  So if you remove the
cell, grow it in culture,

304
00:26:13,000 --> 00:26:17,000
it goes on to become these things
called fibroblasts which are cells

305
00:26:17,000 --> 00:26:21,000
that spread out in the dish.
They don't fuse.  They don't make

306
00:26:21,000 --> 00:26:26,000
all these muscle proteins.
And so the cell, although it's

307
00:26:26,000 --> 00:26:30,000
destined to become muscle,
has not yet made the decision to

308
00:26:30,000 --> 00:26:35,000
take that leap of fate.
However, if you wait a few hours,

309
00:26:35,000 --> 00:26:40,000
and we're actually on your handouts
now.  If you wait a few hours and

310
00:26:40,000 --> 00:26:45,000
you go and you remove the cells that
have arisen from C3,

311
00:26:45,000 --> 00:26:50,000
which are divided now.
They're still labeled and you can

312
00:26:50,000 --> 00:26:55,000
distinguish them because it's a very
powerful dye and doesn't diffuse.

313
00:26:55,000 --> 00:27:01,000
So you can remove that same group
of cells a bit later.

314
00:27:01,000 --> 00:27:06,000
And you can then grow those cells in
a culture, in a simple saline

315
00:27:06,000 --> 00:27:11,000
solution.  And lo and behold they go
on, after some hours,

316
00:27:11,000 --> 00:27:16,000
and form multinucleate myotubes.
They go on to form muscle.  So

317
00:27:16,000 --> 00:27:22,000
somewhere between the 32 cell stage
and the stage of a much older embryo

318
00:27:22,000 --> 00:27:27,000
of many hundreds of cells,
the cells have made the decision to

319
00:27:27,000 --> 00:27:32,000
go onto their particular fate.
So something has changed in terms of

320
00:27:32,000 --> 00:27:36,000
the regulatory genes that they're
expressing, presumably,

321
00:27:36,000 --> 00:27:40,000
that allow them to go and become
muscle.  OK.

322
00:27:40,000 --> 00:27:56,000
So let's break this

323
00:27:56,000 --> 00:28:02,000
down a bit more.
And let's ask the question,

324
00:28:02,000 --> 00:28:07,000
as to what inputs are required to
turning on MyoD activation in a more

325
00:28:07,000 --> 00:28:11,000
defined sense?
And the first thing I'm going to

326
00:28:11,000 --> 00:28:16,000
talk about is how the embryo knows
where dorsal is.  OK?  So

327
00:28:16,000 --> 00:28:25,000
dorsal determination.

328
00:28:25,000 --> 00:28:29,000
And what I'm going to tell you is a
very interesting tale of

329
00:28:29,000 --> 00:28:34,000
determinants, of cell autonomous
regulatory factors that are

330
00:28:34,000 --> 00:28:38,000
specifically required to tell the
embryo where the future dorsal or

331
00:28:38,000 --> 00:28:43,000
backside of the embryo
is going to be.

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00:28:43,000 --> 00:28:48,000
And I'll tell you that these
determinants act to stabilize a

333
00:28:48,000 --> 00:28:54,000
particular protein that is a
transcription factor,

334
00:28:54,000 --> 00:29:00,000
and this protein is called
beta-catenin.

335
00:29:00,000 --> 00:29:06,000
And beta-catenin,
once it's stabilized,

336
00:29:06,000 --> 00:29:13,000
so it is a transcription factor,
goes on to activate a set of genes

337
00:29:13,000 --> 00:29:20,000
that are dorsal specific genes,
but not MyoD.  We're not there yet.

338
00:29:20,000 --> 00:29:37,000
Gene.  G.  I cannot spell, can I?

339
00:29:37,000 --> 00:29:41,000
OK.  So let's discuss this using
your handouts.

340
00:29:41,000 --> 00:29:46,000
This is a movie that I was showing
you at the beginning.

341
00:29:46,000 --> 00:29:50,000
Woops.  Let's show it to you again
because I want to point

342
00:29:50,000 --> 00:29:57,000
something out.

343
00:29:57,000 --> 00:30:00,000
All right.  This is a four cell
embryo.  Maybe it's going to be an

344
00:30:00,000 --> 00:30:04,000
eight cell embryo.
Do you see how these embryos have

345
00:30:04,000 --> 00:30:08,000
got some, two cells that are kind of
darkly pigmented and two cells that

346
00:30:08,000 --> 00:30:12,000
are lightly pigmented?
Yeah?  The lightly pigmented cells

347
00:30:12,000 --> 00:30:15,000
are the ones that are going to be
the future dorsal site of the embryo.

348
00:30:15,000 --> 00:30:19,000
OK?  That's where the muscle is
going to come from.

349
00:30:19,000 --> 00:30:23,000
And these cells on the other side,
on the ventral side of the embryo,

350
00:30:23,000 --> 00:30:27,000
that's where the belly is
going to come from.

351
00:30:27,000 --> 00:30:32,000
Now, how does this happen?
Well, it happens, and I've

352
00:30:32,000 --> 00:30:38,000
augmented the diagram on one of your
handouts a bit so you'll have to add

353
00:30:38,000 --> 00:30:44,000
a circle.  It happens because of
movement of these purple dorsal

354
00:30:44,000 --> 00:30:50,000
determinants.  And these purple
dorsal determinants,

355
00:30:50,000 --> 00:30:56,000
as I'll show you, move within the
newly fertilized embryo.

356
00:30:56,000 --> 00:31:01,000
In fact, within about 16 minutes
after fertilization these dorsal

357
00:31:01,000 --> 00:31:06,000
determinants have moved in the
embryo and have told the embryo

358
00:31:06,000 --> 00:31:11,000
where the future dorsal side is
going to be.  And the way they move

359
00:31:11,000 --> 00:31:16,000
is that, in fact,
the early egg and the zygote consist

360
00:31:16,000 --> 00:31:21,000
of two kinds of cytoplasm,
an outer sphere of cytoplasm that's

361
00:31:21,000 --> 00:31:26,000
kind of gelatinous and an inner
sphere of cytoplasm that

362
00:31:26,000 --> 00:31:31,000
is more liquid.
And in this outer gelatinous

363
00:31:31,000 --> 00:31:35,000
cytoplasm sit these purple
determinants.  And when

364
00:31:35,000 --> 00:31:40,000
fertilization takes place these move
by a good angle,

365
00:31:40,000 --> 00:31:44,000
at least 30 degrees relative to
where they were,

366
00:31:44,000 --> 00:31:48,000
and relative to this inner liquid
cytoplasm, which is why I've got

367
00:31:48,000 --> 00:31:53,000
this doted equator there to indicate
that there is a relative movement of

368
00:31:53,000 --> 00:31:57,000
these determinants.
OK?  It's not the whole embryo

369
00:31:57,000 --> 00:32:02,000
that's moving.
It's just part of the embryo that's

370
00:32:02,000 --> 00:32:06,000
moving relative to the other part.
And this movement of determinants

371
00:32:06,000 --> 00:32:10,000
defines the dorsal and the ventral
side of the embryo.

372
00:32:10,000 --> 00:32:15,000
OK.  Here's a movie to show you
that.

373
00:32:15,000 --> 00:32:22,000
OK.  So this is a movie that is

374
00:32:22,000 --> 00:32:27,000
about the first 20 minutes of
fertilization.

375
00:32:27,000 --> 00:32:32,000
And you can see here pigment moving
from one region to another region.

376
00:32:32,000 --> 00:32:35,000
Now, the pigment is not the
determinants.  It just moves with

377
00:32:35,000 --> 00:32:39,000
the determinants.
OK?  So this is an indication that

378
00:32:39,000 --> 00:32:43,000
there is stuff moving,
actually, from the vegetal pole

379
00:32:43,000 --> 00:32:47,000
region up towards this so-called
animal pole region.

380
00:32:47,000 --> 00:32:51,000
So the terms animal and vegetal
pole indicate two different sides of

381
00:32:51,000 --> 00:32:55,000
the radially symmetric egg.
The animal pole is pigmented.

382
00:32:55,000 --> 00:32:59,000
The vegetal pole is not.  And
they're just useful landmarks.

383
00:32:59,000 --> 00:33:03,000
And, again, this is a frog embryo.
Things may be similar in humans,

384
00:33:03,000 --> 00:33:07,000
but we understand much more about
frogs.  So that's why I'm going to

385
00:33:07,000 --> 00:33:11,000
tell you how things work in frogs.
OK.  So there is a manifestation of

386
00:33:11,000 --> 00:33:15,000
these dorsal determinants moving.
These dorsal determinants move by a

387
00:33:15,000 --> 00:33:19,000
really fantastic process.
They move on microtubules.

388
00:33:19,000 --> 00:33:23,000
Remember microtubules?  What part
of the embryo,

389
00:33:23,000 --> 00:33:27,000
what part of the cell were
microtubules part of?

390
00:33:27,000 --> 00:33:33,000
Anyone remember?
Dredge back in your memories.

391
00:33:33,000 --> 00:33:39,000
I heard something.  Yes?  Thank you.
Cytoskeleton.

392
00:33:39,000 --> 00:33:45,000
Yes.  See me for a frog afterward.
So the microtubules are part of the

393
00:33:45,000 --> 00:33:51,000
so-called cytoskeleton.
They are large rods of proteins,

394
00:33:51,000 --> 00:33:57,000
polymers of proteins.  And when the
sperm enters the egg it brings with

395
00:33:57,000 --> 00:34:03,000
it a centriole that is a
microtubular organization center.

396
00:34:03,000 --> 00:34:07,000
And what it does,
in this series of three pictures,

397
00:34:07,000 --> 00:34:12,000
is to take this mass of red
microtubules that are oriented in

398
00:34:12,000 --> 00:34:17,000
every which way,
and it organizes them so they form

399
00:34:17,000 --> 00:34:21,000
these beautiful parallel tracks of
microtubules in the outer cytoplasm

400
00:34:21,000 --> 00:34:26,000
or between the outer and inner
cytoplasm of the egg or of the

401
00:34:26,000 --> 00:34:31,000
zygote, of the fertilized egg.
So the sperm nucleus or the sperm,

402
00:34:31,000 --> 00:34:36,000
excuse me, the sperm centriole is
organizing this microtubular array.

403
00:34:36,000 --> 00:34:41,000
And it's on these tracks of
microtubules that these determinants

404
00:34:41,000 --> 00:34:45,000
move, kind of like train tracks.
And I'll tell you in a moment, it

405
00:34:45,000 --> 00:34:50,000
really is that way.
So, oh, it didn't quite work.

406
00:34:50,000 --> 00:34:55,000
But OK.  So here is a zygote and
here is this protein I've told you

407
00:34:55,000 --> 00:35:00,000
about, beta-catenin.
Now, in the zygote beta-catenin is

408
00:35:00,000 --> 00:35:06,000
phosphorylated.
And that renders it unstable for

409
00:35:06,000 --> 00:35:11,000
various reasons.
And it's also cytoplasmic.

410
00:35:11,000 --> 00:35:17,000
OK? With the action of the
determinants I've been telling you

411
00:35:17,000 --> 00:35:22,000
about, by about the two to four cell
stage, so very soon after

412
00:35:22,000 --> 00:35:28,000
fertilization the beta-catenin on
one side of the embryo,

413
00:35:28,000 --> 00:35:34,000
the side where these determinants
are has been dephosphorylated.

414
00:35:34,000 --> 00:35:38,000
So there is a post-translational
modification.  Dephosphorylated.

415
00:35:38,000 --> 00:35:43,000
And that renders it stable for
various reasons.

416
00:35:43,000 --> 00:35:47,000
It's no longer subject to protein
degradation.  It also allows it to

417
00:35:47,000 --> 00:35:52,000
enter the nucleus.
It allows it to act as a

418
00:35:52,000 --> 00:35:57,000
transcription factor.
So these determinants are changing

419
00:35:57,000 --> 00:36:01,000
the stability and the protein
structure of this beta-catenin

420
00:36:01,000 --> 00:36:06,000
transcription factor.
Beta-catenin is really an important

421
00:36:06,000 --> 00:36:10,000
protein.  This is a wild type frog
embryo.  Two wild type frog embryos.

422
00:36:10,000 --> 00:36:14,000
The head, tail, here's the eye
forming.  In embryos that have been

423
00:36:14,000 --> 00:36:19,000
depleted of beta-catenin you get
these kinds of blobs that have no

424
00:36:19,000 --> 00:36:23,000
dorsal structures at all.
They've got no muscle.  They've got

425
00:36:23,000 --> 00:36:27,000
no nervous system.
Nothing that would normally come

426
00:36:27,000 --> 00:36:32,000
from the dorsal side of the embryo.
So what is the identity of these

427
00:36:32,000 --> 00:36:37,000
dorsal determinants?
And, again, this you have partly on

428
00:36:37,000 --> 00:36:42,000
your handouts.
And the identity of the dorsal

429
00:36:42,000 --> 00:36:47,000
determinants ergo is something that
stabilizes beta-catenin and

430
00:36:47,000 --> 00:36:52,000
dephosphorylates it.
And what these are believed to be

431
00:36:52,000 --> 00:36:57,000
presently are two proteins called
GBP and dsh.  OK?

432
00:36:57,000 --> 00:37:02,000
You don't need to know the names.
It's the principle that's important

433
00:37:02,000 --> 00:37:08,000
here.  But what's very interesting
is that GBP and dsh proteins bind to

434
00:37:08,000 --> 00:37:13,000
another protein called kinesin,
and kinesin attaches or is part of

435
00:37:13,000 --> 00:37:18,000
the microtubule system.
So you get these determinants

436
00:37:18,000 --> 00:37:24,000
attaching to the microtubules,
and as these microtubules polymerize

437
00:37:24,000 --> 00:37:29,000
and have a rotation associated with
them, it's not quite clear they

438
00:37:29,000 --> 00:37:35,000
rotate at this point,
this GBP and dsh are associated with

439
00:37:35,000 --> 00:37:40,000
the microtubules.
And then these two proteins,

440
00:37:40,000 --> 00:37:44,000
GBP and dsh, also called disheveled,
inhibit another protein called GSK3

441
00:37:44,000 --> 00:37:48,000
which is a kinase that
phosphorylates beta-catenin and

442
00:37:48,000 --> 00:37:52,000
renders it unstable.
So you're getting here dorsal

443
00:37:52,000 --> 00:37:56,000
determination.
You can go think about this more

444
00:37:56,000 --> 00:38:00,000
clearly.  Dorsal determination
because you are inhibiting

445
00:38:00,000 --> 00:38:05,000
an inhibitor.
All right?  Complicated?

446
00:38:05,000 --> 00:38:09,000
Yes.  Beautifully understood?
More or less.  This is really the

447
00:38:09,000 --> 00:38:14,000
most beautiful example that we know
of how you get determination of a

448
00:38:14,000 --> 00:38:18,000
particular region of the embryo.
OK.  So one step.  We probably have

449
00:38:18,000 --> 00:38:23,000
about 20 steps to go.
And clearly we're not going to

450
00:38:23,000 --> 00:38:27,000
cover them all so I'm giving you a
little meander through the number of

451
00:38:27,000 --> 00:38:33,000
steps that are involved.
So another step informing muscle,

452
00:38:33,000 --> 00:38:39,000
that I want to tell you about, is
forming a particular kind of cell

453
00:38:39,000 --> 00:38:46,000
from which the muscle will arise.
So not only does muscle arise from

454
00:38:46,000 --> 00:38:52,000
cells that are dorsally located.
Muscle also arises from a cell type

455
00:38:52,000 --> 00:39:01,000
called the mesoderm.

456
00:39:01,000 --> 00:39:06,000
The mesoderm is part of a system of
cells that are the earliest known

457
00:39:06,000 --> 00:39:12,000
cell types in the embryo.
And these are the germ layers.

458
00:39:12,000 --> 00:39:18,000
And the germ layers consist of
mesoderm, and also of two other

459
00:39:18,000 --> 00:39:24,000
cells types called ectoderm and
endoderm.  OK.

460
00:39:24,000 --> 00:39:30,000
And these cell types were
discovered a lot time ago.

461
00:39:30,000 --> 00:39:33,000
They're not differentiated cell
types but they do have different

462
00:39:33,000 --> 00:39:37,000
properties.  They are stepwise along
the way to becoming different things.

463
00:39:37,000 --> 00:39:41,000
The mesoderm,
shown in red here,

464
00:39:41,000 --> 00:39:44,000
gives rise to the muscle,
blood, kidney, heart and various

465
00:39:44,000 --> 00:39:48,000
other things.  OK?
The ectoderm, for example,

466
00:39:48,000 --> 00:39:52,000
gives rise to all of the nervous
system, and the endoderm to the gut

467
00:39:52,000 --> 00:39:56,000
and the lung.  And they get their
names from their position

468
00:39:56,000 --> 00:40:00,000
in the early embryo.
The endoderm is on the inside,

469
00:40:00,000 --> 00:40:06,000
the ectoderm on the outside, and the
mesoderm between the two.

470
00:40:06,000 --> 00:40:11,000
OK.  How does the mesoderm form?
The mesoderm forms through another

471
00:40:11,000 --> 00:40:16,000
complicated system.
It forms through the action of a

472
00:40:16,000 --> 00:40:22,000
transcription factor called VegT.
And this is a maternally expressed

473
00:40:22,000 --> 00:40:27,000
transcription factor.
So we can distinguish maternal and

474
00:40:27,000 --> 00:40:33,000
zygotic effects, genetically
and in development.

475
00:40:33,000 --> 00:40:41,000
VegT is maternally expressed.
That means it is present in the egg.

476
00:40:41,000 --> 00:40:50,000
VegT in term activates in just one
region of the embryo another gene

477
00:40:50,000 --> 00:40:59,000
called nodal.  It's actually a set
of genes.  Nodal is

478
00:40:59,000 --> 00:41:08,000
a secreted factor.
It is actually a ligand and,

479
00:41:08,000 --> 00:41:16,000
therefore, an inducer.  And it is
zygotically expressed.

480
00:41:16,000 --> 00:41:24,000
So another principle, "zygotically
X" for expressed,

481
00:41:24,000 --> 00:41:33,000
and "maternally X" for expressed.
OK.

482
00:41:33,000 --> 00:41:37,000
This is one your handouts.
So we're talking now about the same

483
00:41:37,000 --> 00:41:42,000
time of development that we've been
talking about but a different system

484
00:41:42,000 --> 00:41:47,000
that is working in parallel.
Here is an embryo.  And I've shown

485
00:41:47,000 --> 00:41:51,000
you between the 200 and the 500 cell
stage that has got this VegT

486
00:41:51,000 --> 00:41:56,000
transcription factor present in the
nucleus.  It's a transcription

487
00:41:56,000 --> 00:42:01,000
factor.
And you can find a concentration

488
00:42:01,000 --> 00:42:05,000
gradient of this thing.
In one part of the embryo at this

489
00:42:05,000 --> 00:42:09,000
equatorial region there are low
amounts of it.

490
00:42:09,000 --> 00:42:14,000
Towards the vegetal pole there are
high amounts.  And where you have

491
00:42:14,000 --> 00:42:18,000
this low amount of VegT you get a
band of cells that's going to become

492
00:42:18,000 --> 00:42:23,000
mesoderm.  This mesoderm then goes
on to express this nodal gene which

493
00:42:23,000 --> 00:42:27,000
is a secreted ligand.
And, for those of you who are

494
00:42:27,000 --> 00:42:31,000
familiar with this,
it's a member of something called

495
00:42:31,000 --> 00:42:37,000
the TGF-beta family.
So this gives you another set of

496
00:42:37,000 --> 00:42:43,000
cells to deal with.
And what I'm going to tell you now

497
00:42:43,000 --> 00:42:49,000
is that to think about where the
skeletal muscle comes from we have

498
00:42:49,000 --> 00:42:56,000
to superimpose the two things I've
told you about becoming dorsal and

499
00:42:56,000 --> 00:43:02,000
becoming mesodermal.
So the next thing we need to do is

500
00:43:02,000 --> 00:43:09,000
to talk about the dorsal mesoderm.
And we can write an equation here

501
00:43:09,000 --> 00:43:16,000
where beta-catenin plus nodal makes
dorsal mesoderm.

502
00:43:16,000 --> 00:43:23,000
And I'll tell you that this dorsal
mesoderm has a very special property

503
00:43:23,000 --> 00:43:31,000
and a special name.  It's
called the organizer.

504
00:43:31,000 --> 00:43:36,000
Is the dorsal mesoderm the future
skeletal muscle?

505
00:43:36,000 --> 00:43:42,000
I'm sorry to tell you it's not
really.  There's another step

506
00:43:42,000 --> 00:43:48,000
involved.  And I will tell you in a
moment that the organizer is going

507
00:43:48,000 --> 00:43:54,000
to induce the precursors of the
skeletal muscle.

508
00:43:54,000 --> 00:44:00,000
So let me write this down just to
give you a sense of where we are.

509
00:44:00,000 --> 00:44:04,000
OK.  So let's move on here.
And let's define dorsal mesoderm by

510
00:44:04,000 --> 00:44:09,000
an equation.  And,
in fact, you can define this whole

511
00:44:09,000 --> 00:44:13,000
process by an equation.
But, of course, the genes that have

512
00:44:13,000 --> 00:44:18,000
to be expressed and so on have to be
expressed in a specific temporal

513
00:44:18,000 --> 00:44:23,000
order.  So it's not a simple linear
equation.  It's got a time component,

514
00:44:23,000 --> 00:44:28,000
too.  OK.  So here we have
beta-catenin on the dorsal side.

515
00:44:28,000 --> 00:44:33,000
And we have mesoderm defined by
nodal signaling in a band across the

516
00:44:33,000 --> 00:44:38,000
equator.  And if we superimpose them
we get a region where both of these

517
00:44:38,000 --> 00:44:43,000
factors are in the same group of
cells.  And where both of these

518
00:44:43,000 --> 00:44:49,000
factors are in the same group of
cells we get a special type of

519
00:44:49,000 --> 00:44:54,000
mesoderm formed called the dorsal
mesoderm or the organizer.

520
00:44:54,000 --> 00:45:00,000
The organizer is a very famous
piece of mesoderm.

521
00:45:00,000 --> 00:45:05,000
And it is famous for this reason.
This is a very old experiment that

522
00:45:05,000 --> 00:45:10,000
was done in the 1920s where one
could take a piece of donor tissue

523
00:45:10,000 --> 00:45:16,000
from a donor embryo,
that is the future organizer,

524
00:45:16,000 --> 00:45:21,000
and move it to a host embryo, and
put it on the side of the embryo

525
00:45:21,000 --> 00:45:27,000
where the organizer wasn't.
OK?  So to put it on the ventral

526
00:45:27,000 --> 00:45:31,000
side of the host embryo.
And when this was done,

527
00:45:31,000 --> 00:45:35,000
to the surprise of the investigators,
because they really didn't do the

528
00:45:35,000 --> 00:45:39,000
experiment for this reason,
they found that they got these

529
00:45:39,000 --> 00:45:43,000
conjoined twins.
Conjoined twin embryos.

530
00:45:43,000 --> 00:45:46,000
Here's your host embryo.
And this is a second embryo that's

531
00:45:46,000 --> 00:45:50,000
formed.  And you could look at see
where that piece of transplanted

532
00:45:50,000 --> 00:45:54,000
tissue went.  And when you did you
found it was in a strip along the

533
00:45:54,000 --> 00:45:58,000
back of the embryo,
but most of the second embryo,

534
00:45:58,000 --> 00:46:02,000
the conjoined embryo came from
tissue that was the host

535
00:46:02,000 --> 00:46:06,000
embryo tissue.
In other words,

536
00:46:06,000 --> 00:46:10,000
this piece of transplanted tissue
had organized or induced a second

537
00:46:10,000 --> 00:46:15,000
embryo, including all the skeletal
muscle.  OK?  And including a head

538
00:46:15,000 --> 00:46:19,000
and a brain and so on.
This is a very famous experiment,

539
00:46:19,000 --> 00:46:24,000
very famous piece of tissue.  And
for this experiment the Nobel Prize

540
00:46:24,000 --> 00:46:29,000
was awarded to Hans
Spemann in 1935.

541
00:46:29,000 --> 00:46:33,000
Here are conjoined frog embryos made
by transplanting organizers.

542
00:46:33,000 --> 00:46:38,000
You can see they have two heads and
they're joined at their tail region.

543
00:46:38,000 --> 00:46:43,000
What about humans?  Well, the same
thing is true in human embryos.

544
00:46:43,000 --> 00:46:48,000
Usually identical twin embryos will
split somewhere between the two cell

545
00:46:48,000 --> 00:46:52,000
stage and a very early stage where
there is something called the inner

546
00:46:52,000 --> 00:46:57,000
cell mass.  And you get two
identical embryos made and you get

547
00:46:57,000 --> 00:47:02,000
completely separate embryos made.
Sometimes, very rarely,

548
00:47:02,000 --> 00:47:06,000
you get incomplete splitting of the
embryo.  And the organizer is split

549
00:47:06,000 --> 00:47:10,000
in two in a human embryo,
and you land up with human conjoined

550
00:47:10,000 --> 00:47:14,000
twins.  This is very rare.
The survival of conjoined twins is

551
00:47:14,000 --> 00:47:19,000
very rare.  There are some instances,
and I've put on your handout a

552
00:47:19,000 --> 00:47:23,000
diagram of, these are actual girls
who had been diagramed that were

553
00:47:23,000 --> 00:47:27,000
just like the frogs,
with shared heads, with a shared

554
00:47:27,000 --> 00:47:32,000
trunk and separate heads.
So the processes in frogs,

555
00:47:32,000 --> 00:47:37,000
as far as we know, are very similar
to those in humans.

556
00:47:37,000 --> 00:47:42,000
Now, what I'm going to do because I
have very interesting stuff I'd like

557
00:47:42,000 --> 00:47:47,000
to finish this off,
finish off here, but I see time is

558
00:47:47,000 --> 00:47:52,000
creeping on, is to end off with a
teaser.  And to tell you that the

559
00:47:52,000 --> 00:47:57,000
final step in getting MyoD on is to
activate these things

560
00:47:57,000 --> 00:48:02,000
called somites.
And I'm going to take five minutes

561
00:48:02,000 --> 00:48:06,000
at the beginning of next lecture to
complete this and then we will move

562
00:48:06,000 --> 00:48:09,000
onto our next topic.  So thank you.