VULVA FORMATION IN

Caenorhabditis elegans

Dr. S. Chakraborti
Why study worms?
 “Thus we want a
multicellular organism
which has a short life
cycle, can be easily
cultivated, and is small
enough to be handled in
large numbers, like a
micro-organism. It
should have relatively
few cells, so that
exhaustive studies of
lineage and patterns
can be made, and
should be amenable to
genetic analysis.” --
Excerpts from Proposal Sydney Brenner
to the Medical 2002 Nobel prize in Physiology or
Research Council, 1963 Medicine
 Caenorhabditis elegans
 C. elegans is a small, transparent, free-living nematode.
 It is the first multicellular organism to have its whole genome
sequenced, and as of 2012, the only organism to have its connectome.
 There are two sexes: a self-fertilizing hermaphrodite and a male.
 C. elegans has five pairs of autosomes and one pair of sex
chromosomes. Sex in C. elegans is based on an XO sex-determination
system. Hermaphrodites of C. elegans have a matched pair of sex
chromosomes (XX); the rare males have only one sex chromosome
(XO).
 The developmental fate of every single somatic cell (959 in the adult
hermaphrodite; 1031 in the adult male) has been mapped.
 The genome contains an estimated 20,470 protein-coding genes. About
35% of C. elegans genes have human homologs.
 Used as model organism in the fields of neurobiology, developmental
biology, apoptosis, sleep research etc.
 Anatomy of C. elegans

Anus
Pharynx Intestine (yellow) Rectum
Gonad (pink) Vulva Epidermis

head ~1 mm tail
anterior posterior

Fig. 5.42
Reproduction in C. elegans
 Most C. elegans individuals are hermaphrodites. In
their early development, they are male and the
gonad produces sperm, which is stored for later
use. As they grow older, they develop ovaries. The
eggs "roll" through the region of sperm storage,
are fertilized inside the nematode, and then pass
out of the body through the vulva.
 The male can inseminate the hermaphrodite,
which will preferentially use male sperm (both
types of sperm are stored in the spermatheca).
 When self-inseminated, the wild-type worm will
lay about 300 eggs. When inseminated by a male,
the number of progeny can exceed 1,000.
The C. elegans gonad: an extremely
efficient reproductive system

Fig. 5.42
 Vulva in C. elegans
 The vulva is a structure on the ventral surface of
adult hermaphrodite females of C. elegans.
 It forms the connection between the gonad and the
outside, and comprises
➢ a link to the gonad
mediated by the anchor cell and central cells of the
vulva
➢ a "tube" formed by vulval cells
➢ an opening through the cuticle (the nematode
exoskeleton)
➢ a set of vulva-specific muscles which mediate
opening of the vulva
 How do cell interactions result in the
formation of an organ?

Vulva formation!
 Steps in Vulval Development
 The newly hatched worm (L1) has 12 ectodermal ‘P cells’ along the
ventral midline of the body. They are known as blast cells, which
undergo additional post-embryonic divisions.

 Each ectodermal P cell divides once to

form a neuroblast (Pn.a = Pn
anterior) and an epidermoblast /
ectoblast (Pn.p = Pn posterior).
 The posterior daughters of P ectoblast
cell divisions (Pn.p cells) are positioned
along the ventral midline. The vulva is
derived from these Pn.p
Steps in Vulval Development
 There are 12 Pn.p cells – P1.p to P12.p – each follows a particular
developmental pathway during post-embryonic development.

 Six of these cells (P1.p–P2.p and P9.p–P12.p) fuse with the

surrounding hypodermal syncytium, known as hyp7, soon after
birth.
 P1.p–P2.p and P9.p–P11.p fuse with the hyp7.
 P12.p gives rise to two daughters, one of which (P12.pp) undergoes
apoptotic cell death, the other cell (P12.pa) forms anal hypodermis,
called hyp12.
 The remaining unfused cells, P3.p–P8.p, are termed
vulval precursor cells (VPCs), because all possess
the potential to be specified as vulval cells. Central body
HOX gene lin-39 is involved in this specification.
Steps in Vulval Development
 Although P3.p–P8.p VPCs originate just after birth (during the L1
larval stage, they are immediately arrested in an extended G1
phase.
 The VPCs remain in this quiescent state until the mid-L3 stage,
when they resume division.
 Proper vulval formation requires the precise control of cell division
timing, cell cycle number, spindle orientation, and polarity.
 Many classes of genes and genetic pathways have been found to
regulate these distinct aspects of vulval cell divisions.
Steps in Vulval Development
 The cell connecting the overlying gonad to the VPCs is called the anchor
cell (AC). The AC secretes the LIN-3 protein, a paracrine factor (Similar
to mammalian epidermal growth factor, or EGF) that activates the RTK
pathway. If the AC is destroyed (or if the Iin-3 gene is mutated), the VPCs
will not form a vuIva, but instead become part of the hypodermis (skin).
 The six VPCs influenced by the anchor cell form an equivalence group.
Each member of this group is competent to become induced by the
anchor cell and can assume any of three fates, depending on its proximity
to the anchor cell.
➢ The cell directly beneath the anchor cell divides to form the central
vulval cells.
➢ The two cells flanking that central cell divide to become the lateral
vulval cells, while
➢ the three cells farther away from the anchor cell generate hypodermal
cells.
 However, if the anchor cell (AC) is destroyed, all six cells of the
equivalence group divide once and contribute to the hypodermal tissue.
 If the three central VPCs are destroyed, the three outer cells, which
normally form hypodermis, generate vulval cells instead.
 C. elegans Vulva development
Early larval stage
Anchor cell (AC) Gonad

VPCs
AC Basement membrane
Gonad

P3.p-P8.p are the Vulva

Precursor Cells (VPCs)

Later larval stage

1° and 2° VPCs make

the vulva
3° 2° 1° 2° 3°
3° VPCs are non-vulval
 Cell division and fates of VPCs
 During the L3 stage, a signal from the gonad and signaling among the VPCs
specifies three central VPCs (P5.p to P7.p) to generate vulval cells.
 P3.p and P4.p divide once (in the L3 stage) and the two daughters join the
syncytial hyp7. This is called the 3O (tertiary) fate.
 P5.p divides in the L3 stage to give rise to 7 progeny which form vulval
structures (the vulA, vulB, vulC and vulD cells). Note that the division
pattern is asymmetric (with the vulval-proximal daughter P5.pp only having 3
progeny and P5.pa having 4). This is called the 2O (secondary) fate.
 P6.p divides in the L3 to give eight progeny (four each of vulE and vulF cells).
These cells contact the anchor cell and make the tube from the uterus. The
divisions of P6.pp and P6.pa are symmetrical. This is called the primary or 1O
(primary) fate.
 P7.p divides in the L3 stage to give rise to 7 progeny which form vulval
structures (the vulA, vulB, vulC and vulD cells). Note that the division
pattern is asymmetric (with the vulval-proximal daughter P7.pa only having 3
progeny and P7.pp having 4 – the opposite orientation to P5.p's daughters). This
is also a 2O (secondary) fate.
 P8.p divides once and the two daughters join the syncytial hyp7 [3O (tertiary)
fate].
 Thus, in an intact, wild-type hermaphrodite, the VPCs adopt their fates in a
precise spatial pattern: 3°-3°-2°-1°-2°-3°.
If anchor cell signaling is disrupted, all
VPCs cells adopt a non-vulva fate

anchor
cell

gonad

3° cell 3° cell 3° cell 3° cell 3° cell 3° cell

no vulva
The VPCs have multipotential
Early stage
Anchor cell

gonad

VPCs

Later stage
Anchor cell
gonad

3° 3° 2° 1° 2° 3°

What is causing the VPCs to be different?

 Steps in Vulval Development
 In the fourth larval stage 22 cells (7 vulval descendants from P5.p,
8 from P6.p and 7 from P7.p) of seven types - vulA, vulB1, vulB2,
vulC, vulD, vulE, and vulF, which differ in their patterns of gene
expression, undergo various movements and fusions to make the
adult vulva. The vulval descendants sequentially form seven
distinct toroids, connects to the uterus, and everts as the
hermaphrodite molts to adulthood.
 Anchor cell invasion: the anchor cell extends process to the
centre of the vulF cells and forms a hole in the epidermis.
 In addition, two aspects of uterine development are crucial to
the development of the vulva.
◦ Generation of the anchor cell: The anchor cell is specified from
among two somatic gonadal cells during the end of the L2 stage
(AC/VU decision).
◦ Uterine Patterning: The anchor cell also patterns the developing
uterus, inducing the six pi cells, which generate the utse cell and
uv1 cells. The uv1 cells attach to the vulF cells, while the anchor cell
ultimately fuses with the utse.
Induction in Vulval Development
The development of the vulva in C. elegans
offers several examples of induction on the
cellular level:
A. lin-3 encodes the AC inducing signal
 Mutants at a locus called lin-3 are Vul (vulvaless).
 The lin-3 gene is active in the AC, and lin-3 is thus thought to
encode the AC signal.
 lin-3 has been cloned and encodes a secreted epidermal growth
factor (EGF)- like product.
B. let-23 encodes the receptor for the AC signal
 Most mutant alleles at the let-23 locus cause embryonic lethality.
 Some alleles however are “leaky”, and surviving hermaphrodites
are Vul.
 LET-23 protein is expressed on P6.p as well as P5.p and P7.p.
 let-23 encodes a transmembrane receptor tyrosine kinase with
motifs typical of an EGF receptor and is believed to be the
receptor for the AC signal from lin-3.
 The vulvaless mutations helped define the

Ras pathway

Lin-3/Epidermal Growth Factor (EGF)

Let-23/EGF Receptor

Let-60/RAS
Sem-5/GRB2 Lin-45/RAF

P6.p becomes the primary cell!

 1. Binding of growth factors to receptor
tyrosine kinases stimulates the
autophosphorylation of specific
tyrosines on the receptors.
2. The phosphorylated receptor then
binds to an adaptor protein called
GRB2 which, in turn, recuits SOS (son
of sevenless) to the plasma
membrane.
3. SOS is a guanine nucleotide exchange
factor which displaces GDP from Ras,
subsequently allowing the binding of
GTP (Ras is already anchored to the
1 2 4
plasma membrane by post-
3 translationally added lipids, shown as a
red line).
4. GTP-bound Ras recruits and activates
5 Raf.
5. Raf initates a cascade of protein
phosphorylation by first
phophorylating MEK.
6. Phosphorylated MEK in turn
phosphorylates ERK.
7. Phosphorylated ERK moves from the
cytoplasm into the nucleus
6 8. It subsequently phosphorylates a
7 number of transcription factors,
including the specific transcription
8 factor called Elk-1.
9. Phosphorylated transcription factors
turn on transcription (gene
expression) of specific sets of target
genes.
The activity of Ras is limited by the hydrolysis of GTP back to GDP
by GTPase activating proteins (GAP). Other abbreviations are: MEK
9 = MAPK/ERK kinase, ERK = extracellular receptor-stimulated
kinase, MAPK = mitogen-activated protein kinase. Kinases are
enzymes that add phosphates to molecules using ATP. Mitogens are
factors (such as growth factors) that stimulate cell division.
Induction in Vulval Development
The
The
C. lin-12 & lag-2 mediate the signal:
receptor:
formation of AC cell lag-2
lin-12
(delta)
(notch)
 In wild-type C. elegans
hermaphrodites, two adjacent
cells, Z1.ppp and Z4.aaa, have
the potential to become the
anchor cell.
 They interact in a manner that
causes one of them to become
the anchor cell while the other
one becomes the precursor of
the uterine tissue.
 The choice is made by lateral
inhibition involving
transmembrane uterine
differentiation signalling
molecule - LAG-2 and its
receptor LIN-12, which is
synthesized by both cells.
Induction in Vulval Development
 In loss-off-function Iin-12 mutants, both cells become anchor cells, whereas in
gain-of-function mutations, both cells become uterine precursors.
 Studies using genetic mosaics and cell ablations have shown that this decision is
made in the second larval stage, and that the lin-12 gene needs to function only
in that cell destined to become the uterine precursor cell. The presumptive
anchor cell does not need it.
 During a particular time in larval development, the cell that, by chance, is
secreting more LAG-2 causes its neighbour to cease its production of this
differentiation signal and to increase its production of LIN -12 protein. The cell
secreting LAG-2 becomes the gonadal anchor cell, while the cell receiving the
signal through its LIN -12 protein becomes the ventral uterine precursor cell.
 Thus, the two cells are thought to determine each other prior to their
respective differentiation events.
 When the LIN -12 protein is used again during vulva formation, it is activated
by the primary vulval lineage to stop the lateral vulval cells from forming the
central vulval phenotype.
 Thus, the anchor cell /ventral uterine precursor (AC/VU decision) illustrates
two important aspects of determination in two originally equivalent cells:
1. First, the initial difference between the two cells is created by chance.
2. Second, this initial difference is reinforced by feedback.
Notch Signaling
 The Notch signaling pathway is a
fundamental signaling system used by
neighboring cells to communicate with
each other in order to assume their
proper developmental role.
 The Notch pathway regulates cell
proliferation, cell fate, differentiation,
and apoptosis in all metazoans.
 The core elements of the Notch
signaling system include the
1. Notch receptor (cell surface)
2. Transmembrane ligands (δ in
Drosophila and vertebrates, Lag2 in
C. elegans) and
3. CSL DNA-binding proteins
 Ligand binding leads to cleavage and
release of the Notch intracellular
domain (NICD), which then travels to
the nucleus to regulate transcriptional
complexes containing the DNA-
binding protein.