Lecture 2: Vulva formation Flashcards
Why is C. elegans used as a model organism?
- Simple: 1000 somatic cells, 302 neurones yet still contains all major cell types.
- Easy to grow in the lab: 3-day generation time.
- Small: Can be used for electron microscopy.
- Transparent. Excellent for microscopy.
- Invariant: Cell lineage and nervous system wiring are completely described.
- Genome is completely sequenced.
Why are C. elegans used for genetic studies of development?
C. elegans organisms lend themselves remarkably well to genetic analysis.
• We can look at genetic effects at a single cell resolution.
• We can also study the interactions between mutants to work out genetic pathways.
• Genes defined by mutants can be cloned using transformation rescue.
• Genes can be knocked out by reverse genetics and the effects can be studied.
• Laser ablation permits us to look at the role of individual cells.
How does the vulva form?
The worms can either be hermaphroditic or male.
Hermaphroditic worms can fertilise themselves as they can produce both eggs and sperm.
There are many different cell fates which can occur. Some cell types fuse with the hypodermis, some adhere to the cuticle and some detach from the cuticle.
The anchor cell controls the vulval precursor cells (VPCs). The VPCs give rise to different lineages: primary, secondary or tertiary.
How were laser ablation studies used?
Laser ablation studies can be used to eliminate single cells to determine what will happen to the developing organism.
• If the anchor cell is ablated, then the vulva doesn’t form. It must provide some sort of signal.
• There must be some sort of hierarchy. Even if VPCs are ablated, the cell next to them will become the next tier down in the hierarchy. There is a replacement hierarchy.
How were vulval development mutants used to study the pathway?
There are some viable mutants of nematode worms which can give us clues as to how the development pathway works.
• Muv is a multi-vulva mutation. Dominant GOF mutation.
• Vul is a vuvla-less mutation. Recessive LOF mutation.
• Both of them are mutations in let-60.
• They can also involve other genes such as let-23 and lin45.
• Epistasis experiments showed the order of the pathway.
• Let-60 (GF) ; let-23 (LF) gives the Muv phenotype. This implies that let-23 is further up the pathway. If let-60 is active, let-23 LOF cannot stop it.
• Let-60 (GF) ; lin-45 (LF) gives the Vul phenotype. Lin-45 can stop the let-60 mutation so it is downstream.
• Mutation experiments found the pathway order and genes. These were later found to correspond to specific molecules. These signals give the primary/secondary cell fate.
How does lateral signalling in VPCs occur?
Lateral signalling is what gives rise to the hierarchical nature of the VPCs.
• Primary cells signal to neighbouring cells to adopt the secondary fate.
• Adjacent cells never adopt the primary fate.
• Fate is partially dicatated by proximity to the AC.
• Lateral inhibition prevents adjacent cells adopting the primary fate.
• Lin-12 is the basis of this.
• In a lin-12 (GF), all 6 VPCs adopt a secondary fate.
• In a lin-12 (LF), no VPCs adopt secondary fate.
• Lin-12 encodes the receptor for the signal from P6p.
How does the tertiary cell fate occur?
One mystery was how the tertiary cell fate is formed, which prevents the multivulval form from occurring.
• Lin-15 mutants are Muv so it must normally inhibit the primary/secondary fates while promoting the tertiary fate.
• Mosaic analysis shows that it acts outside the VPCs and in the hypodermis. Must be separate from the Ras pathway.
• It was discovered that lin-15 mutation required the deletion of two loci: lin-15A and lin-15B.
• There are two pathways operating. You need to get a mutation in class A and class B to get the Muv phenotype.
• At first it was believed that there were two separate pathways (this one and the Ras pathway).
• However, it was found that this system prevents inappropriate Lin-3/EGF activity.
Draw the pathway of differentiation.
See notes.
