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We will see one more set of experiments. We have already learned about fate mapping, so in
that lineage tracing. In fate mapping we focused on finding the original cell from which an entire
set of descendants develop. For example, to identify from which primordial cells a given tissue is
derived.
So, here we are going to do a more advanced way of thinking about it, so it will become clearer
as we go through. So, this slide shows you the early embryonic lineage of C. elegans, it focuses
mainly on germline. So, the zygote with one nucleus is called as the P0, it divides into two cells;
one cell is called as AB and another cell is P1 and if you look at the horizontal line, the one on
the left is little longer from this vertical line compared to the other one, so it is not random it is
intentionally drawn that way to indicate that the cell on the left, that is AB, is a larger cell
compared to P1, the smaller cell. So therefore, there is an asymmetry. Some of the cytoplasmic
components get segregated during this process to the posterior side of P0 and therefore they are
inherited only in P1 and not in AB and vice-versa.
In the next division AB divides to give ABa and ABp. So, this small letter a indicates anterior
daughter of AB, p indicates posterior daughter of AB and this might continue dividing like AB

aaa, AB ppp. Each letter indicates one generation. And P1 divides into two cells one is called
EMS another one is called P2. So, these are same nomenclature as you saw in Ascaris embryo
that was observed by Theodor Boveri in his experiments. And then P2 divides as C and P3, again
P3 divides into D and P4. This is a partial lineage; this is just to familiarize with how this lineage
is shown in a diagrammatic way. So, this is called as a lineage tree. So, this kind of lineage tree
exist for all the 959 cells that make up the adult C. elegans body. So, like Ascaris here again the
embryonic cell division is invariant. So, later in the adult stage you might find worms of various
sizes and that is due to the changes in the cell size itself. So, the embryonic cleavage pattern is
invariant in contrast to our cell divisions which are not invariant.
So this lineage tracing was performed by John Sulston. He followed the early embryonic lineage
up to the adult stage. During that he accidentally discovered evidence for apoptosis, he found
that some cells born in a certain lineage always died and he reasoned out that the cells must be
developmentally programmed to die.
So that is how they obtained the first conclusive evidence that apoptosis exists and that is where
then Bob Horvitz figured out the genetic mechanisms, the genes involved and how do they
function in executing apoptotic program and that is the reason he shared the Nobel Prize with
John Sulston. So, John Sulston got Nobel prize for complete lineage of an organism for the first
time. And Sydney Brenner got it for recognizing the importance of this organism.
(Refer Slide Time: 07:04)

So why are we interested in lineage analysis is coming in the next to next slide. So, if you
destroy a specific cell without affecting the other cells during different stages of embryogenesis,
and monitor the consequences of the lost cell, you will be able to define a phenotypic defect at a
single-cell resolution. It helps us to understand the role of a gene present that cells whose
function is required at a given time for the embryo to develop naturally.
Instead of telling in the absence of a gene, the plant will be dwarf; you identify the tissue that
does not develop naturally, where the proliferation is less, and you try to understand why is that
tissue having fewer cells and go back up to a single cell. This is called as precise phenotyping.
Also, this helps in identifying potential cell-cell interaction. For example, if one cell is destroyed
by laser ablation, then what happens to the fate of other neighboring cells? Do they develop
autonomously, or were they dependent on the cell that was destroyed? So, you can discover
potential cell-cell interactions, and then you can also find out the developmental potentials of a
single cell. For example, a given cell might develop into muscle when the normal neighbor is
present, that neighbor could have induced the adjacent cell to develop into a muscle cell, and if
that cell is absent, then muscle cells are not developed.
Similarly, this helps in identifying the default potential of a cell; for example, if you get rid of the
Y chromosome that determines the male development, then that embryo is going to develop into
a female. So then you realize the developmental potential in humans is becoming a female, so
that is the default state. So, you can find out what is the default state of that cell and when does
that cell get committed to a particular developmental path. So, for example, if a neighbor induces
a specific cell to become muscle, when does it induce? Like how early should I ablate that? So,
these are the things you can discover simply by doing laser ablation.
So what laser ablation is? Laser ablation is when we shoot a laser beam on a specific cell under a
microscope mounted on a glass slide. Since the beam size is so small that it can specifically kill
the nucleus of a given cell. This technique is widely used in many model systems, and a variation
of this called laser capture microscopy where you can specifically pinch off a cell from the
tissue. Then you isolate RNA or protein or whatever you want from a single cell and study about
it.

Let us see an example of how all these can be learned through lineage tracing you with the help
of laser ablation.
(Refer Slide Time: 11:26)

So, we are going to learn some of the genetic concepts in the context of making a particular
organ. So, let us say our question is organogenesis, how an organ develops? What are the
signaling pathways involved? When does it gets committed? All aspects of organogenesis. So,
the organ about which we know the most of any organism is the C. elegans vulva. The apparatus
through which it lays out the embryos or mates with a male to get sperm. That is the organ in
which we first found out that ras is not a proto-oncogene. So, people thought that there are
specific genes that are waiting to be activated; therefore, they can cause cancer. Cancer is caused
when a normal gene that should be turned off at a certain point is not turned off, or a gene that
should not be expressed in a specific context gets expressed. And as a result, they end up
stimulating proliferation. So, people realized ras is not an oncogene and is required for normal
development. In its absence, development does not take place. Also, another signaling like notch
delta has been discovered while solving the problem of how a vulva develops. So, therefore we
are going to use vulval development as an example to learn basic genetics.
Let us begin with the vulval lineage itself. Before that, let us familiarize ourselves with the
vulva.

(Refer Slide Time: 13:20)

This image shows a section of the worm; yesterday, I showed you the full worm, so this is
roughly the middle part of the worm that you see here, and where the arrow is pointed is the
pore-like organ through which the embryos come out. So, this is the opening of the uterus. So,
you have two gonadal arms that connect to a central uterus where you have embryos. And the
green-colored tissue is the muscles that help in the contraction, to push the embryo out. So, this
is a pictorial view of the C. elegans vulva. Our focus is on only the vulval muscle cells and the
vulva itself.
So, to get a good view of all parts of the worm in 3D in different sections can be found on the
website called wormatlas.org. Both cartoon and scanning electron micrographs are available on
this website
(Refer Slide Time: 14:51)

So the first image is an SEM image of the pore. This shows the opening through which the
embryo comes, and many structures are seen here. And the second image shows the GFP that is
expressed only in this particular tissue in the pore.
(Refer Slide Time: 15:33)

So this is the lineage diagram for vulva, so in the very early larva stage, you have six cells. These
are called P3, P4, P6, P7, P8. So, these six cells are the ones that are eventually going to make
the entire vulva, and they go through this lineage shown here. P6.p acquires primary fate, P5.p
and P7.p acquires secondary fate, and other cells acquire tertiary fate.

Cell during the early larval stage, get these three different fates and based on those fates, then
they end up following this lineage. So, for example, a cell with the tertiary fate where the
phenotype is still not visible is not going to provide these cells to make the vulva; only the
primary fate cell can do that.
(Refer Slide Time: 16:50)

So, this is the summary of all the experiments. Now let us do a simple experiment. We know that
these six cells are the precursors of Pn.p's. Now in an intact animal, if you follow the lineage of
any one of the six cells, then you will end up finding P3.p is always giving rise to tertiary fate,
and P6.p always gives primary fate. There is no difference in these cells between larvas; even if
you observe multiple times, therefore this lineage follows the invariant pattern. Now, if you
ablate the gonadal cells, then all these six cells will have only the tertiary fate and will not have
these primary and secondary fates. Now, if you leave the gonadal cells intact and if you ablate
the anchor cell, then you get the same effect, none of this Pn.p's will acquire the secondary or
primary fate. Then you do one more experiment where you ablate all gonadal cells leaving the
anchor cell, then you end up finding it is like wild-type.
So, these three put together tell you that it is the anchor cell that induces these cells to acquire
their fate, primarily the primary and secondary fate for these six cells. So, this is how you work
out to a single cell; the anchor cell is the one that induces the primary and secondary fate. This is
also an evidence for cell-cell interaction.

So now if you want to find out what are the different things each one of these six cells can make.
If you ablate P6.p and now its neighboring cells P5.p or P7.p, one of them randomly acquires
primary fate. So, that tells these cells have developmental potential to become primary as well as
secondary. So, Pn.p's are multipotent. So now if you get rid of anchor cell and all the Pn.p's and
see what happens when there is no induction or the default state a default state when the
induction is not there and neighbors are not there what happens to a given cell, so it acquires the
tertiary fate. So, this tells you the tertiary fate is the default or the ground state but given the
induction they can become primary or secondary. So, essentially any one of these six cells can
acquire any one of the three fates depending on the context.
(Refer Slide Time: 20:37)

So that is an example of how lineage analysis is useful. Next is we are going to learn about a
genetic screen, its importance and how it is done. So, here our goal is understanding how vulva is
formed. So, before beginning, I want to mention a fundamental thing. If you look at the DNA
sequence, the order in which the four nitrogenous bases occur holds biological information. But
chemically, they are either purine or pyrimidine; there is no uniqueness in terms of chemistry for
any given sequence. So, as a result, anything that is going to disrupt DNA chemically is not
going to be sequence-specific. So, we need to remember this; for example, when Morgan's group
used x-ray to mutagenesis, they cannot target the chromosome 1 left side first 100 base pairs. So,
therefore, when you are going to mutagenize using any mutagen, you are going to disrupt DNA

randomly. So, depending on the strength of the mutagenesis- the concentration of the mutagen,
and exposure time of the mutagen on a given organism, one will be able to mutate in such a way
that only a small part of DNA is damaged and not the whole of it. For example, if you are using
chemicals like ethyl methane Sulfonate, EMS. If you take more EMS and treat the worms for a
long time, they are going to be dead because there you are going to make large-scale disruption
on the DNA. So, optimize the concentration such that you will probably create one lesion on the
DNA somewhere in one chromosome or the other, not multiple mutations.
So, the effect of mutagen can be followed by the phenotype, for example, whether it is going to
make wrinkled seeds or round seeds. So that is always the readout here. So, therefore here what
will be our readout? Whether the worm can lay embryo or not after treating with the mutagen.
So, we already saw that self-fertilization allows quick recovery of recessive mutations. And in
the slide, the advantages of this self-fertilization are listed—the first two bullets are advantages
of using C. elegans as a model. The second one tells you the number of chromosomes sets
screened is twice the number of F1 progeny. So, that means, if you are going to take a few
10,000 of hermaphrodites, the young adult, and soak it in a solution containing EMS, you are
likely to have mutations taking place in sperm or oocytes.
So therefore, after mutagenesis treatment with the mutagen, when I am going to clone the
worms, I am going to look at the progeny that has effects on oocyte or sperm; it could be either
one, but you will be able to screen for both. Suppose you compare this with an organism where
you have males and females separately. Suppose I am going to take the female and mutagenize.
And in the progeny, I am only having mutations coming through one gamete if there are no
mutations in the other one. So, that is the advantage here, so if I am going to screen 10,000 F1,
what it means is I am screening 20,000 chromosome sets because sperm and egg come from the
same individual. And usually, it is enough if you screen about 5,000 worms like 10,000 gametes
to find the loss of function in any given gene.
Given its genome size and the fact that you can screen twice the number of chromosomes sets
per individual organism, usually screening about 5000 worms is enough to get a loss of function
like lethal or sterile those kinds of phenotypes.

So usually 50 mM EMS is used, and they typically soak it about for a few hours no more than
that, then they wash off the EMS and then put on plates allowing them to feed on E.coli, and then
you collect embryos from them.
(Refer Slide Time: 26:42)

So now, we will see a very complex genetics screen. In the slide, this is an actual screen. So you
take the worms that are wild-type for a given gene involved in vulval development. Since these
worms have a functional vulva, we will assume that the gene is wild-type +/+; it is diploid. Then
after you treat with the EMS and then you allow it to self, meaning hermaphrodites are going to
give their progeny using their sperm and egg. Now let us say your gene of interest is mutated in
one of the two allelic chromosomes, and we call that as m. And in the other one, the same locus
getting mutated is low. So, therefore we write it as m/+. Now, if you clone the F1 worms, in F2,
you will get 25% of the worms not laying any embryos. Here they are going to be bag of
embryos; they cannot lay embryos, so the worm gets filled with embryos. Since the required
factors for the development of the embryo is present within the eggshell, the embryos develop
and hatch as larvae inside the worm uterus itself. When you are going to have a worm, filled with
worms inside, even 1 in 10,000, that worm will stand out.
So that is another important aspect of how to design a screen. The phenotype that you are going
to look for should be very obvious compared to the background wild-type. So, then it is very
easy to score or identify even a very rare mutagenic event if your screen, the readout that you are
going to plan is such that you can readily observe it even amid 1000s of wild-type progeny, that
is an important thing in genetics screen.
So here even among 10,000 F1's, we can identify the plate with a bag of worms, and then you
pick that. So, that was the result of the screen.
(Refer Slide Time: 29:38)

So, they got about 100 mutants in the initial screen where there was a problem in laying
embryos. And when they decided to look closely at it, they found some of them had multiple
vulval primordium as if it is trying to make more than one vulva. And in some of them, there is
no vulva; they could already classify the phenotypes into two. Possibly you isolated multiple loss
of function alleles for a given gene. So these 100 mutant alleles, like you have a hundred groups
of worms where there is a problem with making vulvas, this does not mean you have identified
100 genes involved in it. Some of them maybe 10 alleles for one gene. And maybe in another
gene, you got only one allele, so it is all possible.
So, to identify the genes that got mutagenized, complementation can be done.
So, if you take one of these 100 and then mate it with another group and if the mutation is on the
same gene, then they will be homozygous for the loss of function of that particular gene. So, they
will be vulvaless, but one is a mutation in gene a required for vulval development; another one is

a mutation in gene b required for vulval development. If you have am/a+ and bm/b+, then you
have a wild-type copy for both of them, and that is called trans-hetero. This is complementation.
So, if they complement, then they are two different genes. If they do not, then they are on the
same gene, so, therefore, each mutant having a phenotype is called an allele because whatever be
that locus, there is a wild-type allele, and you got a new mutant allele. So after doing
complementation, some alleles will group into one group; another one will group into another
group, so you call it complementation groups. So each complementation group is essentially
mutations for a given gene or alleles of one gene. So, this is how these scientists came down to
20 different genes. So, one mutagenesis screen and then complementation has found 20 genes in
vulva development.
(Refer Slide Time: 32:57)

So, now let us go further, 20 different genes involved in vulval development. Still, then we are
working not on a difficult organism, so here we know invariant lineage, laser ablation, and we
thought that we can define phenotype to single-celled resolution. So, let us do that, can I now
find out in each one of the 20 where exactly is the problem to start with during the embryonic or
larval development?
So the detailed observation of mutants is essential. So, in the worms that do not have a vulva, we
must identify where precisely the problem happens during larval development.

I would like you to familiarize yourself with Nomarski optics. So, when you have a thin
transparent specimen and allow light to go through, which we call as bright field, many internal
structures would not be readily visible. So, you will not be able to visualize some of the internal
structures and you may not get a three-dimensional perspective to the image when you do that
sort of a microscopy. So, a person name Nomarski came up with a method, so this uses several
lenses and optical components in a microscope such that the differences in the refractive indices
of the different cellular components.
Let us say you know the nucleoplasm has a different refractive index compared to the cytoplasm
or the nuclear membrane or mitochondria and so on. So, by exploiting the differences in the
refractive indices he managed to create a three-dimensional view of an object and that is what we
call as differential interference contrast microscopy, DIC, or Normarski, in honor of the person
who came up with this method. And that is the image you were seeing when I first showed you
worm, that is a DIC image.
So all those images were DIC images. So here it is just a regular light microscopy with no
staining. Still, by exploiting the differences in the refractive index you get subcellular detail as
well as a three-dimensional perspective of the object. So, using that, you can examine what
happens to these six cells. Since we already know that the wild-type has an invariant pattern,
then one of those 20, lin-26, has a vulvaless phenotype, there we find no Pn.p's, none of the 6
were present, which tells you the wild-type function of lin-26 is required for Pn.p production. So
you discovered a gene that is necessary for the Pn.p formation to start with. Then if you look
another one, lin-3; two different complementation groups again, the phenotype is the same,
vulvaless, but when you look at where, when exactly the problem started, then you find the
Pn.p's do not seem to get the acquired fate. So, they all have the default fate. So conclusively,
that shows lin-3 is required for the induced fate.
In no other biological experiment, in one experiment, you come to a conclusion. This is the
power of genetic methods. So, this is how the genes are discovered; after that, you could do
whatever you want; you can crystallize and solve the structure, find out what happens in the
active site or whether it binds to a ligand, etc.