Zebrafish Primordial Germ Cell Migration Flashcards
What are PGCs?
These are the cells that will go on to form gametes.
In zebrafish these precursors are set aside during development specifically for the purpose of producing sperm and eggs, at which point they adopt a specific morphology and gene expression.
How are PGCs determined in development?
The regions of germ plasm (area of the oocyte which eventually is contained within specific cells) that give rise to the PGCs contain specific determinants, crucially vasa and nanos.
These are proteins that are part of a signalling pathway that fates these regions for germ cell differentiation.
How are PGCs produced in drosophila development?
In the drosophila oocyte the germplasm is localised to one end of the structure, because nanos mRNA is localised to the posterior pole of late-stage oocytes due to selective actin-dependent entrapment (Forrest and Gavis, 2003).
As the embryo develops, the somatic nuclei that form around the edge of the cell to produce the first groups of somatic cells are separated from the pole cells that have already formed from the germplasm.
What enabled study of PGC development in zebrafish?
Vasa is the gene that is most commonly associated with the germplasm in drosophila, finding the zebrafish homologue was what enabled the study of zebrafish PCGs to begin.
Wholemount in situ fluorescence hybridisation studies in zebrafish using vasa and nanos mRNA allow the germplasm to be identified and tracked to the cells that they form.
How is the mRNA for vasa and nanos localised in zebrafish development?
The mRNA for these is not found until the first cellular cleavage event, from which point it is found to be localised to the cleavage planes, spreading to form concentrated points at the four poles of the cell.
They begin migration towards the end of epiboly, and by the time the embryo is at the 3 somite stage the mRNA is localised to the area in which the gonads will form.
How is the vasa and nanos mRNA localisation effected in zebrafish development?
At the very beginning of embryogenesis, maternal vasa mRNA in present throughout the embryo, but it is preferentially degraded by the somites and stabilised by the PGC. This has been shown to be dependent on regulation via the 3’-UTR of the mRNA (Koprunner et al, 2001).
How was the RNA localisation method for vasa and nanos identified by Koprunner et al, 2001?
When the gene was modified to include GFP and compared to a control in which GFP was fused with the xenopus globin 3’-UTR, it was shown that the UTR is sufficient for selective degradation in somites and protection/translation in the PGCs. This was confirmed by morpolino experiments.
What does the mode of migration of the PGCs imply about its regulation?
The first clue as to how PGC migration is controlled was by the observation that it occurs in distinct and dicrete steps.
This suggests that there are distant signalling mechanisms responsible for directing their movement.
What guides PGC migration?
Doitsidou et al, 2002 discovered that the migration was guided by a gradient in a particular chemokine; SDF-1 (stromal-cell derifed factor 1) - AKA CXCL12.
This was surprising as chemokines are mainly associated with the immune system rather than development.
How did Doitsidou et al, 2002 first identify SDF-1 as a candidate for the guiding factor in PGC migration?
This was discovered by morpholino screens which showed CXCR4b to be required for proper migration, CXCR4 being a family of chemokine GPCR receptors that allowed the effect to be traced to SDF-1.
What is the expression profile of CXCR4b in PGCs?
PGCs begin expression of CXCR4b at the same time as they begin migration and continue to do so after they reach the regions in which thay will develop into gonads.
How did Doitsidou et al, 2002 confirm that SDF-1 guided PGC migration in WT zebrafish?
They track the expression of the receptor and its ligand using in situ hybridisation. This showed that they were expressed in zones of PGC migration, SDF-1 appearing to mark the pathway of the migrating cells.
Double staining with SDF-1 and PGC markers showed co-localisation, indicating its increased presence in the migration path and hence its role as an attractant.
What did ectopic expression of SDF-1 in the zebrafish embryo show?
The spadetail mutant causes an altered expression pattern of SDF1 expression throughout the embryo, leading to the PGCs migrating to the anterior of the embryo.
Loss of function of the chordino protein, a BMP antagonist, leads to ectopic expression of SDF1 in the posterior of the embryo, causing the PGCs to migrate to that area.
In fact, injection or ectopic expression of SDF1 anywhere in the embryo causes the PGCs to migrate there.
What was observed when elements of the SDF-1 axis were overexpressed or inhibited?
When SDF1 expression was knocked down using antisense morpholinos it led to a dramatic migration phenotype where the PGCs were randomly scattered in the embryo.
This also occurs upon overexpression of SDF1. This overexpression of SDF1 as well as inhibition of PGC CXCR4 expression causes the cells to lose their polarity and morphology, as this largely based around shaping themselves for travel in a particular direction.
What are neuromasts?
In the adult fish the posterior lateral line is a streak of sensory organs called neuromasts than runs down the length of the fish.
These are composed of a kind of hair cell that detects the pattern of water movement around the fish.
How are neuromasts produced during development?
During development, this line of neuromasts is laid down by a migrating primordium than runs from the head of the fish down to the tail, depositing eight neuromasts down the length of the fish.
How is primordium lateral line migration visualised?
Since the lateral line is located just under the skin, it is easy to visualise simply by dyeing the cells in particular with stains such as Yo-Pro1 or DASPEI, but the most useful method of this is use of the CldnB::lynGFP transgenic line (developed by Haas and Gilmour, 2006), in which the lateral line cells express GFP for easy identification and tracking.
This line has dramatically improved our ability to record dynamic events in posterior lateral line primordium migration (pLLp).
What is the structure of the primordium?
The primordium itself is a worm-like cell group that can be split into two halves – the leading end and the trailing end - which have different cellular phenotypes.
What are the cells in the primordium leading end like?
Cells towards the leading end are more mesenchymal in morphology (i.e. flatter) and are responding to Wnt/Lef signalling.
What are the cells in the primordium trailing end like and what is their role?
Those in the trailing end respond to FGF signalling to assume a columnar epithelial cell morphology, meaning they are taller and have prominent apical and basal polarity.
They become centred in circular rosette structures that form into protoneuromasts.
How are neuromasts formed from the primordium?
Protoneuromasts form and continue to travel with the primordium until stabilisation and maturation is triggered by the expression of the helix-loop-helix transcription factor Atoh1a by the central cell in the rosette.
This TF also fates the central cell to become a sensory hair cell. At this time the nascent neuromast stops its cohesive interaction with the neighbouring cells and is deposited.
Once enough new cells have been produced by proliferation at the leading end of the still-travelling primordium a new protoneuromast is generated at the leading end, causing the cycle to repeat
What controls primordium migration?
The migration of the primordium is partly controlled by the same system as the migration of the primordial germ cells; the SDF-1/CXCR4b system.
What are the isoforms of SDF-1?
SDF-1a and b are also known as CXCL12a and b respectively.
What is the limitation of SDF-1 - CXCR4b regulation of primordium migration?
While this does determine the path down which the primordium migrates, it is not thought to be responsible for the directional control, as it expression is uniform down the lateral line at any given time.
