caenorhabditis elegans (c. elegans) an elegant worm

45
Caenorhabditis elegans (C. elegans) An elegant worm

Upload: aubrie-mcdowell

Post on 13-Jan-2016

236 views

Category:

Documents


2 download

TRANSCRIPT

Page 1: Caenorhabditis elegans (C. elegans) An elegant worm

Caenorhabditis elegans (C. elegans)An elegant worm

Page 2: Caenorhabditis elegans (C. elegans) An elegant worm

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

Page 3: Caenorhabditis elegans (C. elegans) An elegant worm

C. elegans: the chosen one!

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

Short generation time: 3 days

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

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

Page 4: Caenorhabditis elegans (C. elegans) An elegant worm

Life cycle of C. elegans

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

Page 5: Caenorhabditis elegans (C. elegans) An elegant worm

Anatomy of C. elegans

Pharynx Intestine (yellow)Gonad (pink) Vulva

RectumAnus

Epidermis

head tailanterior posterior

~1 mm

Fig. 8.43

Page 6: Caenorhabditis elegans (C. elegans) An elegant worm

Hermaphrodite (XX)

Males (X0)

Hermaphrodites do it by themselves

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

Page 7: Caenorhabditis elegans (C. elegans) An elegant worm

The C. elegans gonad: an extremely efficient reproductive system

Fig. 8.42

Page 8: Caenorhabditis elegans (C. elegans) An elegant worm

Movie of C. elegans development

Page 9: Caenorhabditis elegans (C. elegans) An elegant worm

Within this lineage is the secret of embryonic development

John Sulston

Page 10: Caenorhabditis elegans (C. elegans) An elegant worm

All neural synapses have been mapped

Page 11: Caenorhabditis elegans (C. elegans) An elegant worm

Learn to read a lineage diagram!

= Cell death

Line ending = differentiated cell

Branching = cell division

Increasing age of worm

1st stage larva

2nd stage larva

embryo

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

allowing one to follow the cell lineage.

Page 12: Caenorhabditis elegans (C. elegans) An elegant worm

Cleavage Events

Lineage

P0 zygote 2 cell stage 4 cell stage 8 cell stage

Page 13: Caenorhabditis elegans (C. elegans) An elegant worm

Most lineages consist of multiple tissue typesbut the P4, E and D cells gives rise to a single tissue type

Fig. 8.43

Page 14: Caenorhabditis elegans (C. elegans) An elegant worm

Mutations can alter lineages in many ways

Page 15: Caenorhabditis elegans (C. elegans) An elegant worm

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?

Page 16: Caenorhabditis elegans (C. elegans) An elegant worm

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

Page 17: Caenorhabditis elegans (C. elegans) An elegant worm

Even cell death is programmed into the lineage

C. elegans was used to identify the machinery that regulates

programmed cell death in vertebrates

Page 18: Caenorhabditis elegans (C. elegans) An elegant worm

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

Page 19: Caenorhabditis elegans (C. elegans) An elegant worm

Partitioning of cytoplasmic determinants

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

P0

AB

P1

P3

P4

blue nuclei green P-granules

P granules are asymmetricallysegregated into one cell, the P4 cell, which will give rise to the germline

Page 20: Caenorhabditis elegans (C. elegans) An elegant worm

Movie of P-granule movement

Page 21: Caenorhabditis elegans (C. elegans) An elegant worm

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

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

Page 22: Caenorhabditis elegans (C. elegans) An elegant worm

Moss E. 2007. Current Biology, R425.

wildtype lin-14 (lof) lin-4 (lof)

Lof= loss of function, gene function is disrupted

Timing of developmental events

Lin-14 is required for the timing of cell division in the L1 stage. Lin-4 regulates transition from L1 to L2 stage..

Page 23: Caenorhabditis elegans (C. elegans) An elegant worm

L1 L2 L3 L4 Adult

LIN-4

LIN-14

Lev

els

Time

wildtype lin-14 (lof) lin-4 (lof)

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

Page 24: Caenorhabditis elegans (C. elegans) An elegant worm

L1 L2 L3 L4 Adult

LIN-4

LIN-14

Lev

els

Time

wildtype lin-14 (lof) lin-4 (lof)

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

If you have a mutation that results in an INCREASED level of LIN-14 (gain of function) which lineage would you expect

Page 25: Caenorhabditis elegans (C. elegans) An elegant worm

lin-4 does not encode a protein—what????

It encodes for a microRNA

lin-4

lin-14

lin-4

Translation blocked!

lin-14lin-4

Page 26: Caenorhabditis elegans (C. elegans) An elegant worm

Cell-Cell Interactions: the P2 impact!

Apx-1/Delta-like ligand

Glp-1/Notch receptor

mom-2/Wnt ligand

mom-5/Wnt receptor

Signal from P2 cell required to induce EMS cell to produce E cell which forms the gut (see p. 248)

Page 27: Caenorhabditis elegans (C. elegans) An elegant worm

How to cell interactions relate to the formation of an organ?

Vulva formation!

Page 28: Caenorhabditis elegans (C. elegans) An elegant worm

Getting the terminology down: C. elegans Vulva

Figure 6.27Anchor cell (AC) Gonad

VPCs

Early larval stage

P3.p-P8.p are the Vulva Precursor Cells (VPCs)

AC Basement membrane

Gonad

Later larval stage

P5.p,P6.p and P7.p lineages make the vulva

P3.p,P4.p and P8.p lineages non-vulval

1° 2°2° 3°3°P6.p P7.pP5.pP3.p P4.p P8.p

Page 29: Caenorhabditis elegans (C. elegans) An elegant worm

Inductive and lateral signals induce the vulva

gonadAnchor cell

VPCsP3 P4 P5 P6 P7 P8

VPCs after induction 1° 3°3° 3° 2°2°

The primary and secondary cells form the vulva

Page 30: Caenorhabditis elegans (C. elegans) An elegant worm

How’d you know that? Cell ablation studies helped identify key players in vulva formation

Lecture notes: experiment 1

Page 31: Caenorhabditis elegans (C. elegans) An elegant worm

Movie of cell ablation

Page 32: Caenorhabditis elegans (C. elegans) An elegant worm

gonad

anchorcell

3° cell3° cell 3° cell

If anchor cell signaling is disrupted, all VPCs cells adopt a non-vulva fate

3° cell 3° cell3° cell

no vulva

Page 33: Caenorhabditis elegans (C. elegans) An elegant worm

The VPCs have multipotential

gonad

Anchor cell

1° 2° 2° 3°3° 3°

What is causing the VPCs to be different?

Early stage

Later stage

gonad

Anchor cell

VPCs P3 P4 P5 P6 P7 P8

Page 34: Caenorhabditis elegans (C. elegans) An elegant worm

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

gonadAnchor cell

VPCs P3 P4 P5 P7 P8

1° 2° 2° 3°3° 3°

2° 2° 3°3° 3°

A

3° 3° 3°3° 3°

B

2° 1° 2°3° 3°

C

P6

Lecture notes: experiment 2

Page 35: Caenorhabditis elegans (C. elegans) An elegant worm

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

Page 36: Caenorhabditis elegans (C. elegans) An elegant worm

Inductive and lateral signals induce the vulva

gonadAnchor cell

VPCsP3 P4 P5 P6 P7 P8

VPCs after induction 1° 3°3° 3° 2°2°

The primary and secondary cells form the vulva

Page 37: Caenorhabditis elegans (C. elegans) An elegant worm

Lin-3/Epidermal Growth Factor (EGF)

Let-23/EGF Receptor

Sem-5/GRB2

Let-60/RAS

Lin-45/RAF

P6.p becomes the primary cell!

The vulvaless mutations helped define the Ras pathway

Page 38: Caenorhabditis elegans (C. elegans) An elegant worm

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

signal

(VPC cells)

LIN-3

Another representation of the RAS pathway

Page 39: Caenorhabditis elegans (C. elegans) An elegant worm

The Ras mutation is so prevalent that kits are available to test of mutations that are linked to cancer

Page 40: Caenorhabditis elegans (C. elegans) An elegant worm

A signal from P6.p actives notch (lin-12) in P5.p and P7.p

Figure 6.27

Page 41: Caenorhabditis elegans (C. elegans) An elegant worm

Both membrane and receptor are membrane bound!

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

“ Primary cell”

“ Secondary cells”

Page 42: Caenorhabditis elegans (C. elegans) An elegant worm

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

Figure 6.28

The signal:lag-2 (delta)

The receptor: lin-12 (notch)

Page 43: Caenorhabditis elegans (C. elegans) An elegant worm

Does the Notch pathway remind you of anything you learned earlier?

No notch=neural!

Page 44: Caenorhabditis elegans (C. elegans) An elegant worm

Nervous system

Epidermis

Extra nervous system

No epidermis!

Some cells become neuroblasts and signal their neighbors to

remain epidermis

If signal is missing...

all cells eventually ingress and become neuroblasts

The story of epidermal vs. neuronal fate in Drosophila

Page 45: Caenorhabditis elegans (C. elegans) An elegant worm