Stem Cell Regulation by Niche Derived Signals Flashcards
How does the drosophila ovary niche assemble?
• Next step in ovary development is formation of terminal filaments
• Right at the anterior tip of each germarium is the terminal filament – a linear arrary of cells
• Several of these form across the developing ovary, and each will define the location of one ovariole
• The terminal filaments express Delta the membrane ligand for Notch.
− Notch is expressed on the somatic precursor cells
− If they receive a signal from Delta, they will differentiate into a Cap cell
− There are normally around 5-6 Cap cells recruited to each terminal filament
→ Delta also marks intestinal stem cells
→ So common signals are being deployed in different functions
How do GSCs recruit to the cap cell niche?
• Happens in a late larval to pupal transition
• As niche formation betweens, the 140 GPCs divide into two populations
1. GSCs in contact with the niche (Bag of marbes –ve cells)
2. Cells which differentiate directly (Bag of marbles +ve cells)
• It is the Bam -ve cells that are recruited to the niche to form stem cells.
What determines which GPCs become the GSCs in the ovary?
• At larval to pupal transition, around 4-7 GPCs are partitioned into each germarium
• But only 2-3 become GSCs
• There are 2 possible models
1. All larval GPCs have equal potential to become GSCs. GPCs are randomly selected to contact the Cap cells and adopt a GSC fate
2. GPCs are already non-equivalent, and a subset of them are pre-determined to adopt the GSC fate.
How can we tell which?
Ashoka and Lin, 2000:
• Constructed transgenic drosophila lines that express GFP in the posterior pole cells that become germline progenitors
• Transplanted an individual GFP expressing pole cell into a non-GFP expressing recipient
• Can then follow the fate of the germ cell as it develops.
• Record the position of the GFP +ve GPC (anterior or posterior)
• Allow it to grow to adult and determine if the ovary GSCs are GFP +ve
➢ The anterior located embryonic GPCs are predetermined to become GSCs in the adult ovary because all of the germline is expressing GFP
➢ With posterior located GPCs, where the GFP +ve cell has not ended up a stem cell, only the early cysts have GFP, not the full germline lineage.
• So, before the niche has been formed, cells are already pre-determined as to which will become the GSCs and which will form the first few eggs of the adult female
1. When the Cap-cell niche is formed, you have homing of pre-determined GPCs with GSC potential to the niche
2. The GSCs establish and become anchored to the niche by cadherin cell junctions
3. The remainder of the germline differentiate into cystoblasts rather than GSCs.
How can you detect lost stem cells from a niche?
Detecting lost stem cells from a niche:
• In one experiment, one week after mutant clone induction, a negatively marked cyst must be the product of a negatively marked stem cell
• But in this germarium, both GSCs are lacZ positive → therefore, the lacZ negative cell that is inferred to have been present must have been lost.
• So now we have an assay for the frequency of loss of GSCs from the niche
How was the role of cadherin in anchoring cells to the niche detected?
- If you tried to make a homozygous null mutant, you would never reach the adult stage
- You cant study its function in the heterozygous state, because it is recessive, so you wont see the phenotype
- You can only see the phenotype if you have both copies
- To overcome this, you use the mitotic clone technique
- One chromosome has the cadherin mutant
- The homologous chromosome is wild-type, but has a lacZ gene
- Chromosome duplication and strand switching can produce two daughter cells, one of which is negatively marked for b-gal and one of which is homozygous for b-gal → the cadherin mutant is the negatively marked one.
- This way we can generate GSCs homozygous mutant for cadherin
- Can use this assay to see how frequently negatively marked cysts (which must have lost their cadherin) occur.
➢ In wild-type marked cells, there is a very slow decline in the population over weeks
➢ In both cadherin and beta-catenin (the intracellular binding partner of cadherin) mutants, there is a very rapid decrease in the frequency of these GSCs.
➢ → This means that cadherin is required for keeping the stem cells in the niche
- If you do microscopy, you can see accumulations of cadherin at junctions between the GSC and Cap cells
- This suggests stem cells are retained in niches by cell—cell adherans junctions.
How does anchorage to the drosophila ovary niche compared with other identified niches?
eg) Drosophila testis
• Instead of Cap cells, have Hub cells
• Usually find at least 3 GCSs adhered to Hub cells via cadherins junctions
eg) The HSC niche
• Cadherins junctions mediate adhesion between HSCs and osteoblasts
eg) Drosoophila follicle somatic stem cells
• There are two kinds of junction in this niche:
1. Integrins mediate contact to the underlying ECM
2. Cahderins mediate contact with adjacent escort cells (Transient support cells)
• These stem cells are kept in a vary specific location
• If you compromise either of these two contacts, you compromise the retention of cells in the niche
eg) Drosophila intestinal stem cells
• Stark contrast to the follicle somatic stem cell
• Here, we purely have an ECM contact
• No specific cellular location for the niche – the stem cells are scattered through the tissue, as there is basement membrane and ECM all throughout the tissue.
What is the role of DPP and Bam signaling in the Drosophila ovary?
Role of DPP Signalling in the Drosophila Ovary
• Overexpression of DPP leads to tumour-like expansion of spectrosome containing germline SCs.
• In the wild-type, GSCs undergo asymmetric division to produce one copy of themselves, and one cystoblast – but with DPP, it just undergoes self-renewal
• Expression of DPP is therefore sufficient to trigger copious self-renewal
DPP is part of the BMP signaling pathway
- DPP ligand binds to the receptors
- This recruits Mad – the drosophila homologue of Smad
- Mad is phosphorylated by the receptor, causing the recruitment of the Drosophila co-Smad homologue Madea
- This complex then moves to the nucleus to regulate gene transcription – one of these is Dad
- -> reduction in DPP signaling results in failure to maintain GSCs
- -> GSC mutant cells defective in the receipt of DPP signal divide more slowly
- -> reduction in the ability to receive DPP signal leads to decreased lifespan of stem cell
What is the DPP signaling doing? How does it effect differentiation of the GSCs?
–> Bag of marbles (Bam) expression is switched on in cystsleaving the niche:
–> DPP signaling is restricted to cells close to the niche:
• Use Dad/lacZ reporter → lacZ expressed when Dad expressed
• DPP is expressed highest in those cells closed to the Cap cell niche
• In cystoblasts it is starting to go down
• In the cysts it has gone
- So there is reciprocal relationship – where DPP is signaling, Bam isn’t expressed
- Might propose that DPP signaling is turning off Bam expression
Test:
• Use temp. sensitive mutants of DPP – displays the phenotype at 29C, but WT at 18C
• Grow flies at 18C to get them through development – then switch to 29C as adults
• In the DPP mutant, Bam is starting to switch off in these cells
• Overexpression of DPP in the niche represses Bam expression
• The Bam mutant phenotype resembles the phenotype of DPP overexpression → accumulation of 1000s of GSC-like cells
• Overexpression of Bam rescues the consequences of overexpression of DPP
→ so Bam is required to repress GSC fate.
So the model is:
• DPP causes expansion self-renewal of the germline stem cells to maintain their population by repressing Bam expression.
• Bam represses the GSC fate.
Compare signals that maintain the Drosophila ovary SC population with those of other niches
eg) Drosophila testis niche
• Unpaired – Drosophila ligand of JAK-STAT pathway expressed in Hub cells
• Unpaired signal via JAK to maintain GSC self-renewal
eg) Drosophila intestinal niche
• Wingless (Wnt) growth factor diffuses across the ECM from adjacent circular muscle cells
• Wnt overexpression explands the population of ISCs
eg) Drosophila follicle somatic stem cell niche
• Hedgehog is a diffusible GF originating in the somatic cells at the anterior of the germarium
• Hedgehog maintains follicle somatic stem cells
eg) The GSC niche in C.elegans
• Delta signaling via Notch maintains the GSC population
