L10 : Primordial Germ Cells and Chemokine Signalling Flashcards
What are primordial germ cells (PGCs)?
Precursors to sperm and eggs, set aside during development
- Distinctive morphology and ‘germ plasm’
- Specific gene expression
Note: drosophila and zebrafish use this mechanism
Where do zebrafish PGCs come from?
Specified early in development and marked by several key genes, which assist in maintaining PGC identity
What are 2 key marker genes for zebrafish PGCs?
Vasa
Critical early marker gene for PGCs and active role in germ cell development
Nanos
Transgenic lines have been created using 3’ UTR
- Restricts reporter expression to PGCs
- Allows live visual tracking
What controls PGC migration?
Signalling mechanisms enable distinct PGC migratory steps
SDF1 chemokine signalling via Cxcr4b receptor is controlling migration of PGCs
What were the key investigations into control of PGC migration and what did they show?
- Expression analysis of SDF1/Cxcr4b
- Analysis of defective migration in morphants by timelapse
- Gain of function study (overexpression of SDF1)
What did expression analysis of Cxcr4b/SDF-1 show?
Analysed by in situ hybridisation
- Expressed in zones of PGC migration
- Double staining with PGC markers demonstrates colocalisation
- SDF1 lines of expression appears to mark pathway for guiding migration
What did analysis of defective migration by live imaging in knockdown and overexpression show?
Live imaging of migration using GFP reporter (driven by nanos promoter)
- Knockdown of SDF1 and Cxcr4b (inject morpholino antisense RNA to prevent translation)
- Guiding chemokine signal removed - Overexpression of SDF1
- Non-localised signals throughout embryo
Results show PGCs end up ectopically located
- PGCs lose directionality (polarity) and fail to reach gonad
What is the lateral line?
Comprised of sensory organs called neuromasts (hair cells) down the side of fish
Like hearing underwater
How does the lateral line develop?
From large primordium that migrates down side of embryo
Rosette maturation followed by deposition
About 8 neuromasts produced in first wave
What is the organisation and signalling pathways during lateral line development?
Migrating cell population divided into 2 zones
Leading zone:
- Wnt signalling is active
- Pathway promotes cell migration and proliferation, maintaining undifferentiated state
Trailing zone:
- FGF (fibroblast growth factor) signalling active
- Pathway regulates cell adhesion and patterning
- Promotes epithelial organisation and deposition
What controls migration during lateral line development and how is this known?
SDF1/Cxcr4b
Cxcr4b mainly active in leading zone, controlling migration
Tracking SDF1a expression:
- Defective expression seen in fss mutant causes defective migration
- Primordium may U-turn or drop down to migrate along with primordial germ line
What is the role of Cxcr7 in lateral line primordium and how is this known?
Cxcr7 expressed in trailing zone
- Ligand sequestration
- Helps establish and maintain the chemokine gradient that directs migration
Chemokine flood
- Used method to flood chemokines
- Initial slow migration then recovery
- Dependent on Cxcr7 in posterior zone
What are common chemokine signalling features between PGCs and lateral line primordium?
- Both use SDF1/Cxcr4b signalling to guide migration
- Both employ Cxcr7 to shape gradient and direction of migration
- Both involve migration over long distances
How is chemokine signalling linked to cancer progression?
Tumour cells can exploit chemokine signalling pathways to move and invade new tissues
- Several metastatic cancers have resemblance to lateral line guided migration
- Angiogenesis (formation of new blood cells) is driven by chemokines
Examples of why large scale genetic screens are useful in zebrafish?
- Can elucidate roles for genes
- Wnt/PCP pathway in gastrulation
- Chemokine gradient generation by cells/tissue
Examples of why transgenic markers are useful in zebrasfish?
- Easy to create/integrate
- Label specific cell populations (lateral line, PGCs, gastrulating cells)
- Read out of pathway activity
- Study gene regulation (eg. through nanos 3’ UTR and enhancer/promoter regions)
