2. Primordial germ cells

Question 1

What are the stages of primordial germ cells becoming sperm and eggs?

Answer 1

Migrate
Divide
Differentiate

Question 2

What are primordial germ cells?

Answer 2

Specialised cells that give rise to the germline
Are central to reproduction and continuation of the species

Question 3

What are the 3 similarities between PGCs in different organisms?

Answer 3

1. Ability to generate haploid gametes by meiosis
2. Gene expression profiles e.g. Vasa and Nanos are highly conserved
3. PGCs arise generally very early in development (despite not needing them until an adult)

Question 4

What is the difference between preformation and epigenesis mechanisms of PGC specification?

Answer 4

Preformation = inheritance of maternal determinants consiting of ribonucleoparticles which become localised to one location of the oocyte and several cells in the developing embryo which give rise to PGCs in adult.
Epigenesis = induction from ‘unspecialised cells’ and requires zygotic paracrine signalling - No pre-PGC factors in the oocyte, some cells in the zygote signal to other cells and tell them that they are going to become PGC

Question 5

Describe PGC specification in drosophila.

Answer 5

First cells to form
Germ plasm becomes sequestered at posterior pole of oocyte during oogenesis.
Hierarchy of factors needed to assemble germ plasm including Oskar (seems to be master localiser) and Vasa.
If mislocalise Oskar to anterior, get anterior PGCs (make trans-gene, put on 3’ UTR of a gene that is expressed at the anterior, the UTR will be sufficient to get the trans-gene to mislocalise at the anterior, dragging other components of the germ plasm with it and forming PGC at the anterior end.)

Question 6

What is the germ plasm responsible for in drosophila?

Answer 6

Localisation and translation of maternal RNAs and proteins as well as their protection from degradation.
A mode of cellularisation that is specific to PGCs
Global transcriptional silencing on PGCs (zygotic genome is shut down until after the onset of gastrulation)

Question 7

Describe PGC specification in C. elegans.

Answer 7

Germ line i set by series of asymmetric partitioning of maternal P granules during early cell divisions.
P1 has all P granules and divides giving rise to a somatic cell and P2 cells which again has all P granules > repeat until P4 which gives rise to ferm cells.
Germline blastomeres inherit cytoplasmic RNA-rich organelles called P granules and transcriptional repression mediated by maternal PIE-1 which blocks complex responsible for phosphorylating RNA polymerase II

Question 8

Describe PGC specification in Zebrafish.

Answer 8

Germplasm becomes localised to distal ends of cleavage furrows.
Gets segregated into 4 cells which divide to give about 30 PGCs.
PGC fate maintained through germ plasm RNA-binding protein dead end (Dnd)
Dnd Knock down embryo PGCs respond to cues in their environment by differentiating into a range of cell types. Role is conserved in many other vertebrate species

Question 9

Describe PGC specification in Mouse

Answer 9

Originate from subset of cells located in proximal epiblast.
Requires BMP signalling from surrounding extraembryonic ectoderm (4 and 8b) and visceral endoderm (BMP2) and Wnt3 from posterior visceral endoerm and epiblast.
Induce formation of PGC precursors identifed by expression of transcriptional repressor BLIMP1.
Wnt signalling may be important to make the epiblast competent to respond to BMP signalling for PGC specification.
Antagonistic signals from anterior visceral endoderm prevent induction of PGCs in anterior epiblast.
Initial 6-8 BLIMP1 positive cells proliferate to give about 40 PGCs by E7.25

Question 10

Describe postembryonic germline specification.

Answer 10

Segmented annelid worm Capitella teleta: Germline normally arises from a single cell in the 64-cell embryo. If you ablate that cell, most offspring will lack PGCs in larvae, but you see large diffuse upregulation of vasa/nanos positive somatic cells and adults mostly fertile. Ability of somatic cells to dynamically acquire germline fate during postembryonic development, despite stereotypes germline fate acquisition in only one cell during embryogenesis

Planarian flatworms Schmidtea mediterranea: Capable of regenerating any lost body part including germline (in sexual strains). Somatic stem cells, neoblasts, serve as the source for all new adult tissues. After amputation to remove all PGCs, a subset of neoblasts expressing nanos can give rise to new PGCs. For males, require dmd-1 positive somatic cell so cell signalling important.

Question 11

What is the expansion of the fittest theory of the evolution of preformation?

Answer 11

Organisms specifying PGCs using germ plasm meant that there was disentanglement of germ-line specification from somatic influences – i.e. BMP signalling in mouse required for specification of mouse could be free to evolve and be used for something else in development if mouse evolved to use preformation for specification of PGCs.
“liberation” of the selective pressures on somatic gene regulatory networks.
Networks were less constrained and could accumulate more heritable mutations (protein sequence evolution).
This could allow diversification of body patterns and allow species to evolve faster into empty ecoscapes.
Difficult to test and contested in literature.
Greater number of species and species diversity correlated in clades that have preformation (frogs, birds, teleosts) compared to those that have epigenesis (turtles, salamanders, lancelets)
Species richness alone might be a poor indicator of speciation or diversification rates because it doesn’t include the role of clade age, generation time, birth-death rates, reproductive isolation.

Question 12

What is the timing of PGC formation theory of preformation evolution?

Answer 12

Early developmental timing of PGC establishment, rather than liberation of gene regulatory networks, that drives species evolvability.
Mouse and salamander both use epigenesis.
Mouse PGC specification occurs before gastrulation, but salamanders occur afterwards.
Know there are a lot more species of mouse.
Mice might therefore exhibit release of selective constraint on somatic GRNs, rapid evolution (enhanced evolvability), and increased speciation, like what is predicted for organisms with germ plasm. Whereas others, such as salamanders, evolve slowly.
The fact that mouse PGCs are specified so early, it may relieve any constraints on development.

Question 13

Describe eukaroytic chemotaxis and movement

Answer 13

LArge cells with receptors on cell surface allows for directed cell migration where the cell is always moving in the right direction.
Cell can tell that more of its receptors are occupied on one specific side and if it needs to find the source of the chemoattractant it needs to move in that direction.
Creates polarity - leading and lagging edges
Moves in direction of leading edge up the concentration gradient (reverse if molecule is chemorepellant)

Question 14

Describe bacterial chemotaxis and movement.

Answer 14

“Run and tumble mechanism”
In the absence of any signal, flagella are swimming. At some point there’ll be an event where the flagella flat out (tumble) then bundle up again and swim (running).
If there’s no gradient, periods of running are similarly timed but doesn’t really go anywhere. If going up a chemotaxic gradient, it suppresses that tumbling event. Bacteria isn’t always migrating in the correct gradient.
Bacteria are very small so by just being static, they can’t detect a chemotaxic gradient – not enough surface area to detect the difference between what’s at the front and what’s at the back

Question 15

Describe cell migration using actin-based leading edge protrusions

Answer 15

Lamellipodium sent out at leading edge.
Lagging side has myosin contraction which squeezes cell and pushes contents of cell forward into newly formed area at the leading edge. Has to form new adhesions at the front to substrate and release it at the back.
Want actin depolymerisation at the lagging edge and actin polymerisation at the leading edge.
Complex regulators that control actin cytoskeleton.
Arp complex promotes actin branching and incorporation of new monomers

Question 16

Describe PGC migration in mice

Answer 16

Move prom posterior primitive streak, through hindgut to embryonic endoderm where they become motile.
Migration into genital ridges is dependent on guidance (SDF1 ligand and CXCR4 GPCR) and survival (steel ligand and Kit receptor tyrosine kinase) pathways.
Genital ridge is mesenchyme of mesonephric origin.
On arrival, PGCs undergo morpholigical changes such as loss of polarised morphology and motility. PGCs are now termed gonocytes, proliferate and start their sex-specific development

Question 17

Describe PGC differentiation in males mice.

Answer 17

Become stem cells called gonocytes
Cyp26b1 expression in testes blocks RA signalling, thereby preventing male germ cells from initiating meiosis.
Cyp26b1 expression decreases, Nanos2 is expressed and prevents meiotic initiation by blocking Stra8 expression. Induces male-type differentiation in the germ cells. Meiosis initated after bith

Question 18

Describe PGC differentiation in female mice.

Answer 18

Proliferate and form oogonia.
RA secreted from mesonephros reaches gonad and triggers meitoic initiation via the induction of Stra8 TF.
Meiosis initiated at E13.5 (earlier than in males)
Developing oocytes enveloped by ovarian somatic cells from foetal gonads. Oocyte releases paracrine factors to instruct these support cells

Question 19

Describe PGC migration in zebrafish

Answer 19

Respond to signals from intermediate targets on the way.
Specified at 4 different locations, randomly positioned with respect to developing embryo axis.
At the end of gastrulation, most PGCs accumulated in 2 medial-to-lateral lines at the head trunk border - rest align along lateral trunk mesoderm borders in more posterior regions.
During early somitogenesis, both group of cells migrate towards lateral mesoderm of anterior trunk. Represents intermediate target for PGCs as it isn’t precursor for gonad.
PGCs divide on average 3 times during migration

Question 20

Describe cell migration using blebs and cytoplasmic flow

Answer 20

Live imaging reveals no actin in leading edge protrusions. At leading edge, there’s some release of actin before hydrostatic pressure pushes cytoplasm into the leading edge, creating blebs that are initially low in actin

Question 21

Where does CXCR4b act?

Answer 21

Expressed both in germ cell and somatic
1) expression patterns can be informative in this particular case but doesn’t really answer the question of if its cell-autonomous (required in a neighbouring somatic cell to make the germ cell migrate) or not because it has such high expression
2) chimeric animals through transplantation:
a) transplantation - WT germ cells into cxc4b mutants (reach target), cxcr4b mutant germ cells into WT (mismigration) – so was required in the germ cells themselves
b) genetically – use specific drivers to replace gene function in limited cell types; make homozygous mutant cell clones in otherwise heterozygous background

Question 22

How does CXCR4-SDF1 signalling link to PGC polarity?

Answer 22

CXCR4b uniformly distributed around plasma membrane. Binding activates scaffold protein lrsp53 and promotes formation of dynamic filopodia at cell front. Theory that lot of filopodia forms a lot of SA for the receptors which means more places for ligand to bind > greater receptor-ligand populancy at leading edge > increases polarity (positive feedback).
Local elevation of pH which requires carbonic anhydrase 15b.
Rac1 activity higher at higher pH which therefore promotes actin polymerisation. Front inhibits other parts of the cell from assuming front characteristics

Question 23

Describe how expression of SDF-1a guides germ cells via intermediate targets.

Answer 23

Between 1 somite and 12 somite stages, see germ cells always associated with SDF-1.
Patches of cells express and secrete SDF-1  germ cells go up the concentration gradient there to meet those intermediate targets  move along to eventually meet their final destination.
Either final point secretes more SDF-1 to get the germ cells to leave intermediate point to get to final destination or SDF-1 somehow disappears.
Don’t see obvious gradient at mRNA level using in situ hybridisation.

Other GPCRs can bind SDF-1
Injected oligonucleotide that blocks expression CXCR7  very strong mismigration phenotype
CXCR7 not expressed by primordial germ cell
Important but not expressed in the germ cells themselves
Can bind and internalise SDF-1.
In CXCR7 mutant, doesn’t fully downregulate SDF-1 in intermediate target so some germ cells get stuck there and don’t make it to the final destination

Question 24

Describe PGC migration in drosophila

Answer 24

Start at posterior pole, pushed into embryo through gastrulation, into midgut - bilateral migration through mesoderm - gonad formation.
Regulated by Hmgcr pathway - expressed in somatic cells, leads to germ cell attraction
pathway that forms cholesterol in humans but forms lipids that modifies proteins in flies and synthesises hormones

Regulated by lipid phosphates and Hedgehog.
Lipid phosphate phosphatase – enzymes sitting at cell surface, have extracellular active site and can phosphorylate extracellular lipid phosphates including phosphoric acid, lysine
phosphoric acid and sphingosine phosphoric acid
Hedgehog is secreted signalling molecule and binds to receptor on germ cell

Question 25

How is PGC migration linked to extragonadal germ cell tumours

Answer 25

PGCs migrate in humans Most germ cell tumours (GCTs) originate in testis and occur in adolescents and young adults but 3% of GCT cases are extragonadal and occur in neonates and infants May localise in almost every structure along the midline of the body Assumed these arise from stray PGCs which have undergone a malignant transformation after arresting migration in the midline during embryogenesis

Question 26

Describe in vitro generation of PGCs in mice

Answer 26

Start with mouse ESCs (pluripotent, derived from ICM of preimplantation embryos) and iPSCs. Create epiblast-like cells Under appropriate conditions, able to generate PGC-like cells. Used as donor cells to mice lacking endogenous PGCs showing these were capable of gametogenesis in vivo – sperm were capable of fertilisation and gave rise to healthy offspring Not been successful in any other species until recently when method reported in rats but the step from PGC to sperm requires an in vivo niche. Can use transplantation back into gonads or co-culturing with dissociated mouse gonad somatic cells, but high variability and low scalability Derived foetal ovarian somatic cell-like cells in vitro from murine pluripotent embryonic stem cells, using a stepwise, directed differentiation strategy which resembled in vivo differentiation Used morphogens to stimulate signalling pathways that guide the differentiation of mouse pluripotent cells via an intermediate state Granulosa- and stromal-like cells, as well as less mature precursors, were generated, with profiles of gene expression that closely resembled their counterparts in vivo When such FOSCLCs were co-cultured with PGCLs the “reconstituted ovarioids” supported follicle formation Could be in vitro fertilised and transplanted to produce healthy offspring but as in all of these types of studies, efficiency issues and use of transgenic constructs

Question 27

Describe in vitro generation of PGCs in humans

Answer 27

Use of reporter constructs e.g. BLIMP1-tdTomato Know we have PGCs because of transcriptional profiling vs non-human primate PGCs

Question 28

Describe generation of human oogonia from iPSCs in vitro.

Answer 28

To initiate sexual differentiation, combined hPGCLCs with somatic cells from mouse foetal ovaries to form 3d organoid-like aggregates, called xenogeneic reconstituted ovaries and cultured them for 17 weeks Organoids underwent morphological changes akin to those occurring during human ovary development in vivo Genome-wide gene expression and DNA methylation analyses indicate that the differentiating hPGCLCs resembled human ovarian germ cells at a stage before their entry into meiosis.