Specification and early development of the germ cell lineage

Dr Andy Childs

Department of Comparative Biomedical Sciences, The Royal Veterinary College

achilds@rvc.ac.uk

@DrAndyChilds

Extavour & Akam (2003) Development

Transmit genetic information to the next generation

Maintain genetic integrity across generations

Generate of genetic diversity through meiosis

Generate highly-specialised haploid gametes

Re-programme the genome to enable successful embryonic development after fertilisation

Why study germ cells?

How are germ cells formed?

How do they get to the gonads?

What determines whether germ cells become sperm or eggs?

Outline

How are germ cells formed?

pre-formation vs epigenesis

Strome & Lehmann Science 316:392

Pre-formation: germ cell specification in Drosophlia

Egg is a single large cell with single nucleus

Germ plasm (red) is actively transported to the posterior pole of the embryo before fertilisation

PGCs first cells to be formed – areas containing germ plasm bud off fromembryo

Remaining soma undergoes nuclear divisions without forming new cellsGerm plasm: a mix of mRNA and

proteins required to specify germ cellsIncluding Dazl, Vasa, Nanos, Piwi

Transplantation experiments reveal germ plasm alone is sufficient to specify germ cells

Extractgerm plasmandinject it intoanterior pole

Germ cells format both poles

Germ cells format posterior

pole

Works in flies, worms, frogs and zebrafish – all use pre-formation

Illmensee & Mahwohold (1974):

• Germ cells in mammals appear relatively ‘late’ in embryonic development (after many cell divisions)

- ~6d after fertilisation in mouse

- ~4wks after fertilisation in humans

• Mammalian embryos have no obvious germ plasm

• In mammals, germ plasm components (Nanos, Vasa, Dazl) are important for germ cell development, but not specification

• Germ cell formation in mammals is ‘inductive’ i.e. germ cells are induced to form in response to specific signals

• - also known as ‘epigenesis’

Pre-formation of germ cells doesn’t happen in mammals

Studying primordial germ cell specification in mammals is tough

Events happen just after embryo implantation• enclosed within mouse uterus – can’t observe in real time

Germ cells don’t grow when isolated and cultured

1954-2002 only marker of PGCs was alkaline phosphatase• comes on at e7.5 – PGCs already specified and migrating by then

2 major advances in last fifteen years:• identification of new genes that mark PGCs before e7.5

• development of transgenic mice expressing fluorescent proteins under the control of PGC-specific gene promoters

allows real-time tracking of germ cells in cultured embryos

Germ cell specification in mammals is dependent on signals from extraembryonic tissues

EPIBLAST

ANTERIORVISCERAL

ENDODERM(AVE)

EXTRA-EMBRYONIC ECTODERM (ExE)

Bmp4Bmp8b

Inhibitory signals ensurePGCs form on correct side ofembryo

PGCs

BMP4 and BMP8b from extra-embryonic tissues are are required for PGC specification

Ying Y et al. Molecular Endocrinology 2000;14:1053-1063Bmp8b expression in

Extra-embryonic ectoderm

As with Bmp4, embryos lackingBmp8b fail to form PGCs

Fragilis: marks all cells in the proximal epiblast with potential to become germ cells

Stella: switched on by cells with highest levels of Fragilis

Stella is the earliest marker of ‘true’ primordial germ cells (PGCs)

Fragilis and Stella are the earliest markers of Primordial Germ Cell (PGC) precursors

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Early PGCs switch on germ cell genes and switch off somatic (body) cell genes

Germ

cell

genes

Som

ati

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ll g

enes

Primordial germ cells Somatic cells

The master regulator of germ cell development is probably a repressor of gene transcription

Germ

cell

genes

Som

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Blimp1: a good candidate?

• Blimp1 was already known as a transcriptional repressor in lymphocyte development

• Expressed at the right time – just before Stella

• Group of six epiblast cells, that sit closest to the extraembryonic ectoderm

• these cells may get the highest dose of Bmp4?

Blimp1: expressed in the right place, right time to be the master regulator of PGC fate

EXTRA-EMBRYONIC ECTODERM (ExE)

EPIBLASTBlimp1 PGC precursor cells (arrowheads) in the proximal

epiblast – initially 4-8 cells at e6

Ultimate founders of the PGC lineage

Blimp1 stops PGC precursors from adopting a somatic cell fate

Blimp1 (PRDM1) PRDM14

PGCs

Germ

cell

genes

Som

ati

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ll g

enes

Blimp1 WTPGCs

Somatic cells

Blimp1 KOPGCs

In the absence of Blimp1, PGC precursors express somaticcell genes, and expression of PGC genes is variable

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Blimp1 works in tandem with Prdm14 and Tcfap2c to specify primordial germ cells

Blimp1

Prdm14

Tcfap2c

Germ cell Gene expression

Somatic cellGene expression

Germ cell Migration

How do the germ cells get to the gonads?

PGC migration – back into the embryo

E7.5PGCs moveFrom extra-embryonic

allantois to endoderm

of theembryo

E8 – E9.5As gastrulationOccurs, the Endoderm moves inside the embryo to form the hindgut.Migrating PGCs are carried along with it.

Nature Reviews Molecular Cell Biology (2010) v11 p37-49

PGC migration – from hindgut to gonad

E9.5From the

hindgut, PGCsmove

laterallyInto the

mesenchyme Of the

embryo

E9.5 – E11.5Finally the PGCs move from the mesenchyme out to the developing gonadal ridges.Migration is complete by E11.5.

PGCs increase in number as they migrate 40 at e7.5 to 25,000 by e13.5

http://php.med.unsw.edu.au/embryology/index.php?title=Primordial_Germ_Cell_Migration_Movie

Time lapse videos of mouse PGC migration

What guides migrating PGCs to the gonads?

WT – germ cells tightly arranged in gonads

KO – germ cells dispersed throughout the fetus

Proc Natl Acad Sci U S A. 2003 Apr 29;100(9):5319-23. Epub 2003 Apr 8.

Receptor on PGCs, Sdf-1 made by somatic cells along route

In zebrafish and chick embryos, Stromal Derived Factor 1 (Sdf-1) acts as a chemo-attractant for PGCs, directing their migration. Does the same hold true for mouse?

Stromal Derived Factor 1 (Sdf-1) provides a directional cue for PGC migration to the gonads

No Sdf1 added – PGCs colonise gonads in embryo culture

Addition of Sdf1 – PGCs fail to colonise because signal from all directions swamps production from gonad

Germ cells lacking the Sdf-1 receptor (CXCR4) fail to colonise the gonads efficiently

BUT: a lot still make it and early migration OK – other factors? De

velo

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Number of PGCs reaching the gonads reduced in KO

WT KO

Spermatogenesis or Oogenesis?

What determines whether a germ cell becomes a sperm or an egg?

Gonadal environment determines germ cell fate, not the genetic sex of germ cells

Ad

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At e11.5, XY andXX PGCs can be

sex reversed

At e12.5, only XX PGCs can be sex reversed

At e13.5, PGCs ofBoth sexes arefate restricted

RetinoicAcid

Cyp26B1

Nanos2

Fgf9

A balance of signals determines the fate of a germ cell

Female fate

(oogenesis, meiosis,follicle formation)

Male fate

(Spermatogenesis,germ cell arrest)

PGC

OvaryTestis

Germ cell arrest is accompanied by changes in expression of cell cycle regulators

Loss of phosphorylationof pRB – blocks cell cycle

Increase in levels of cellcycle inhibitor p16INK4a – blocks phosphorylation of pRB

↑germ cell arrest

Germ cell arrest in the testis is accompanied by maturational changes

16wk human fetal testis

As germ cells enter mitotic arrest, they down-regulate stem cell markers (e.g. Oct4, Stella, Blimp1)

‘Mature’ germ cell markers (e.g. Dazl, Vasa) are switched on.

Testicular germ cell cancer cells show retained or reactivated expression of stem cell markers (e.g. Oct4) – failed maturation?

OCT4VASA

In the developing ovary, mitosis continues, forming germ cell nests

Pepling M E Reproduction 2012;143:139-149© 2012 Society for Reproduction and Fertility

Mouse fetal/neonatal development (days)

Nests are connected by intercellular bridges, that allow sharing of cytoplasm and organelles

KIF23 and TEX14 are protein components of the cytoplasmic bridges between oocytes

Biol Reprod. 2009 March; 80(3): 449–457.

Nests enable germ cells to synchronise their divisions, and their entry into meiosis

Pepling and Spradling Development 1998

DividingGerm cells

Nest breakdown releases oocytes to associate with somatic cells and form follicles

Oogonial nests

Mitosis and Meiotic prophase I

Primordial FollicleFormation

Meiotic arrest

Nest breakdown

Association of oocytes with somatic cells

OOCYTES NOT ASSEMBLEDINTO FOLLICLES DIE

FOLLICLES ARREST UNTIL PUBERTY

What causes germ cell nests to break down and form follicles?

Primordial Follicle

Oogonial Nest

Required for nest breakdown:NoboxFiglaFoxl2Wnt4Bmp4

Inhibiting nest breakdown:Estrogens*

ActivinKit ligand?TNFalpha

SCP1

See : Mol. Hum. Reprod. 2009;15:795-803

Massive loss of oocytes occurs during fetal life

Adapted from Vaskivuo, 2002

Germ

cell

nu

mb

er

Follicle formation happens against the backdrop of meiosis

Nature Reviews Genetics 13, 493-504 (July 2012)Nature Reviews Genetics 13, 781-794 (November 2012)

Human oocytes can remain arrested for >50 years

Why do so many oocytes die?

Nature Reviews Molecular Cell Biology 2, 838-848 (November 2001)

Downhill all the way? Oocyte numbers decline steadily from birth.

Kelsey T et al. Mol. Hum. Reprod. 2012;18:79-87

Neo-oogenesis: creating new oocytes after birth?

Postnatal neo-oogenesis in mammals

Neo-oogenesis: the production of new oocytes throughout the adult life of the organism

• Source of debate throughout the early 20th C

• Zuckerman (1950s) – developed a new method of counting oocytes in the ovary– concluded that no more are produced after birth

Postnatal neo-oogenesis does happen in some mammals

Loris tardigradus – red slender loris Nycticebus coucang– greater slow loris

Postnatal oogenesis in mice and humans?

• Johnson et al (2004) challenged the dogma:

• Counted atretic (dying) oocytes across the first three months after birth

• Concluded that the number of atretic oocytes was so high (16-33% at d40/42) that the ovary would run out of oocytes in early adulthood

• So.. something must be replacing them…?

The first controversyJohnson J et al (2004) Germline stem cells and follicular renewal in the postnatal mammalian ovary Nature 428:145-150

Vasa-positive germ cells at surface of ovary. Some of these incorportate BrdU into their DNA – undergoing mitosis

Vasa staining (brown) Vasa staining (green)BrdU mitosis marker (red)

Grafting experiments – proof that oogonial stem cells exist?

Graft GFP ovary onto wildtype (non-GFP) ovary.

If stem cells in donor GFP ovary make new oocytes, they will be GFP-positive and should mix with non-GFP host granulosa cells and make a mixed follicle

Problems with the first study

• OK…

if 16-33% of the oocyte population is dying at d42, there must be 60+ new oocytes made every day

These need to go through meiosis to replace the ones that are dying, to maintain a ‘steady state’

• So where are they?

Germ cells in the bone marrow?• Johnson J et al (2005) Oocyte generation in adult mammalian ovaries by

putative germ cells in bone marrow and peripheral blood Cell 122:303-315

WT

KO+ BMTKO+ BMT

But… Bone Marrow transplants can restore fertility after chemotherapy…

Lee H et al. JCO 2007;25:3198-3204

Low toxicity chemotherapy High toxicity chemotherapy

Isolated oocyte stem cells from the ovary generate live mice when injected back into the ovary

Immunomagnetic bead sorting for Mvh

Long term culture

Injection into ovary gives rise to Oocytes and live mice

Zhou et al (2009) Nat Cell Biol 11:631-36

Mouse and human OSCs produce oocyte-like cells spontaneously in culture

White et al (2011) Nature Medicine 18:413

Oocyte stem cells (OSCs) give rise to oocytes and embryonic structures (maybe..)

White et al (2011) Nature Medicine 18:413

Germ cells in mammals are formed by epistasis

Germ cells must migrate to the gonads, directed by chemical signals

Germ cells have the capacity to adopt a male or female fate, depending on environment

In the fetal testis, germ cells enter mitotic arrest until after birth

In the ovary, germ cells form nests, enter meiosis and form follicles, which remain dormant until puberty

Summary – fetal germ cell development

McLaren A (2003) Primordial Germ Cells in the Mouse. Developmental Biology v262 p1-15

Saitou M and Yamaji M (2010) Germ cell specification in mice: signaling, transcription and epigenetic consequences.

Reproduction v139 p931-942

Tingen C, Kim A and Woodruff T (2010) The primordial pool of follicles and nest breakdown in the mammalian ovaryMolecular Human Reproduction v15 p795-803.

Bowles J, Koopman P (2010) Germ cell sex determination in mammals: extrinsic versus intrinsic factors.

Reproduction 136:943-58

Further reading