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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
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
Life cycle of C. elegans
Photo credit: http://www.scq.ubc.ca/genetic-studies-of-aging-and-longevity-in-model-organisms/
Anatomy of C. elegans
Pharynx Intestine (yellow)Gonad (pink) Vulva
RectumAnus
Epidermis
head tailanterior posterior
~1 mm
Fig. 8.43
Hermaphrodite (XX)
Males (X0)
Hermaphrodites do it by themselves
Photo credit: http://homepages.ucalgary.ca/~dhansen/worms.gif
The C. elegans gonad: an extremely efficient reproductive system
Fig. 8.42
Movie of C. elegans development
Within this lineage is the secret of embryonic development
John Sulston
All neural synapses have been mapped
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.
Cleavage Events
Lineage
P0 zygote 2 cell stage 4 cell stage 8 cell stage
Most lineages consist of multiple tissue typesbut the P4, E and D cells gives rise to a single tissue type
Fig. 8.43
Mutations can alter lineages in many ways
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?
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
Even cell death is programmed into the lineage
C. elegans was used to identify the machinery that regulates
programmed cell death in vertebrates
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
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
Movie of P-granule movement
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
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..
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
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
lin-4 does not encode a protein—what????
It encodes for a microRNA
lin-4
lin-14
lin-4
Translation blocked!
lin-14lin-4
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)
How to cell interactions relate to the formation of an organ?
Vulva formation!
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
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
How’d you know that? Cell ablation studies helped identify key players in vulva formation
Lecture notes: experiment 1
Movie of cell ablation
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
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
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
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 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
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
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
The Ras mutation is so prevalent that kits are available to test of mutations that are linked to cancer
A signal from P6.p actives notch (lin-12) in P5.p and P7.p
Figure 6.27
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”
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)
Does the Notch pathway remind you of anything you learned earlier?
No notch=neural!
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
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