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Caenorhabditis elegans (C. elegans) An elegant worm. Why study worms?. Sydney Brenner.

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slide2

Why study worms?

Sydney Brenner

“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 to the Medical Research Council, 1963

slide3

C. elegans: the chosen one!

Easily cultivated: can grow thousands on a petri dish, feed on non-hazardous bacteria, and cheap to maintain

Short generation time: 3 days

Small: 1 mm (about the size of a pinhead)

Few cells: The adult has 959 hermaphrodrodite (XX) or 1031 (XO) cells

Amenable to genetic analysis: maintained as hermaphrodites, but males exist for genetic studies,The genome is small- 100 Mb

Transparency: allows for development to be analyzed from a single cell and all cells to be lineage

Photo credit: Ian D. Chin-Sang (Queen's University, Kingston, ON, Canada).

life cycle of c elegans
Life cycle of C. elegans

Photo credit: http://www.scq.ubc.ca/genetic-studies-of-aging-and-longevity-in-model-organisms/

slide5

Anatomy of C. elegans

Anus

Pharynx

Rectum

Intestine (yellow)

Epidermis

Gonad (pink)

Vulva

head

~1 mm

tail

anterior

posterior

Fig. 5.42

slide6

Hermaphrodites do it by themselves

Hermaphrodite (XX)

Males (X0)

Photo credit: http://homepages.ucalgary.ca/~dhansen/worms.gif

slide8

Within this lineage is

the secret of embryonic development

John Sulston

slide10

An entire C. elegans hermaphrodite worm consists of exactly 959 cells EVERY SINGLE TIME,

allowing one to follow the cell lineage.

Learn to read a lineage diagram!

Branching =

Increasing

cell division

age of worm

embryo

1st stage

larva

2nd stage

larva

Line ending =

differentiated cell

= Cell death

slide11

P0 zygote

2 cell stage

4 cell stage

8 cell stage

Cleavage Events

Lineage

Fig. 5.42

slide12

Most lineages consist of multiple tissue types

but the P4, E and D cells gives rise to a single tissue type

Fig. 5.42

slide14

Question:

1.) How many cell divisions took place in the wildtype lineage? ____

2.) In wild-type, how many total descendants will cell A have? ____

3.) How many differentiated cells from the wild-type lineage will be a part of the adult worm? ____

4.) What is the best description of the defect in mutant 1?

slide15

How are the invariant lineages established?

ie. How do cells know who they are and what they are doing?

  • Control of apoptosis
  • Partitioning of cytoplasmic determinants
  • Timing of developmental events
  • Cell-Cell interactions
slide16

Even cell death is programmed into the lineage

C. elegans was used to identify

the machinery that regulates

programmed cell death in vertebrates

See also p. 96 for further discussion of cell death pathways

slide17

The Nobel Prize in Physiology

or Medicine 2002

"for their discoveries concerning ’

genetic regulation of organ development

and programmed cell death'"

Sidney Brenner

H. Robert Horvitz

John Sulston

slide18

Partitioning of cytoplasmic determinants

P-granules (green) are cytoplasmic determinants that are formed from ribonucleoprotein complexes that specify the germ cells

blue nuclei

green P-granules

P3

P0

P granules are asymmetrically

segregated into one cell, the P4 cell, which will give rise to the germline

P1

AB

P4

Fig. 5.44

slide19

PARtition mutants (PAR) disrupt the asymmetric distribution of p-granlues

Photo credit: http://mbg.cornell.edu/cals/mbg/research/kemphues-lab/images/par_phenotypes.gif

slide20

Timing of developmental events

Lof= loss of function, gene function is disrupted

lin-4 (lof)

lin-14 (lof)

wildtype

Moss E. 2007. Current Biology, R425.

Lin-14 is required for the timing of cell division in the L1 stage.

Lin-4 regulates transition from L1 to L2 stage.

.

slide21

lin-4 (lof)

lin-14 (lof)

wildtype

Graph of LIN-14 and LIN-4 levels in a wildtype embryo

LIN-4

Levels

LIN-14

Time

L1

L2

L3

L4

Adult

slide22

If you have a mutation that results in an INCREASED level of

LIN-14 (gain of function) which lineage would you expect

lin-4 (lof)

lin-14 (lof)

wildtype

Graph of LIN-14 and LIN-4 levels in a wildtype embryo

LIN-4

Levels

LIN-14

Time

L1

L2

L3

L4

Adult

slide23

lin-4 does not encode a protein—

It encodes a microRNA !!

lin-4

lin-4

lin-14

lin-4

lin-14

Translation blocked!

slide24

Cell-Cell Interactions: the P2 impact!

GLP-1

Notch receptor

APX-1

Delta-like ligand

MOM-2

Wnt homolog

MOM-5

Frizzled homolog

Signal from P2 cell required to induce EMS cell

to produce E cell which forms the gut (see p. 198-199)

c elegans vulva development
C. elegans Vulva development

Early larval stage

Figure 3.34

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° VPCs are non-vulval

inductive and lateral signals induce the vulva
Inductive and lateral signals induce the vulva

Anchor cell

gonad

P4

P7

P8

P3

P5

P6

VPCs

VPCs after induction

The primary and secondary cells form the vulva

slide29

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

The VPCs have multipotential

Anchor cell

gonad

P8

P6

P3

P7

P5

P4

VPCs

Later stage

Anchor cell

gonad

What is causing the VPCs to be different?

slide31

Let’s do an experiment: what happens when the P6.p cell is ablated?

Anchor cell

gonad

P8

P3

P7

P5

P4

VPCs

A

P6

B

C

Lecture notes: experiment 2

slide32

What genes specify the VPC cell fate?

Looked for mutants that disrupted vulva formation

1) No vulva: worms hatch inside (yuck!!)

1) Too many vulvas

Lecture notes: experiment 3

inductive and lateral signals induce the vulva33
Inductive and lateral signals induce the vulva

Anchor cell

gonad

P4

P7

P8

P3

P5

P6

VPCs

VPCs after induction

The primary and secondary cells form the vulva

slide34

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!

slide35

The Ras pathway is abnormally activated in many human tumors

eg: pancreatic cancer, colorectal cancer, lung adenocarcinoma, gall bladder cancer, bile duct cancer and thyroid cancer

Another representation of the RAS pathway

(VPC cells)

LIN-3

signal

slide37

The transmembrane receptor is the Lin-12 protein, a receptor protein related to Notch

“ Primary cell”

“ Secondary cells”

Figure 3.33

Both membrane and receptor are membrane bound!

slide38
Generation of Different Cell Types From Equivalent Cells in C. elegans:Initial specification of the Anchor Cell also requires Notch

The signal:

lag-2 (delta)

The receptor: lin-12 (notch)

Figure 3.35

slide40

The story of epidermal vs. neuronal fate in Drosophila

If signal is missing...

Some cells become neuroblasts

and signal their neighbors to remain epidermis

all cells eventually ingress and become neuroblasts

Nervous system

Extra nervous

system

Epidermis

No epidermis!