The Germline and Reprogramming

Question 1

What are primordial germ cells?

Answer 1

PGCs are the first germ cells. 3-5 PGCs can populate the entire lineage. They’re found at the back end of the embryo in extra embryonic tissues, having got there through the primitive streak (mice). In humans they’re found in the yolk sac

Question 2

How do PGCs travel to their final resting place?

Answer 2

They migrate into the developing gut and head north until they reach the developing gonads (genital ridges). They exit the hindgut and colonise the genital ridges where they continue to develop;

Question 3

How is the sex specified?

Answer 3

Gonadal somatic cells in male embryos turn on SRY (on Y chromosome) and this triggers a downstream cascade that leads to male gonadal and germ cell development. In females SRY is not expressed so female pathway proceeds

Question 4

What happens when you force expression/delete SRY?

Answer 4

Forcing expression of SRY in XX embryos, they develop testis but are infertile. If SRY is deleted in XY embryos, they develop ovaries

Question 5

What are the 3 main processes in the PGC stage of germ cell development?

Answer 5

Specification (induction of PGCs), migration (to the genital ridges), epigenetic reprogramming (sex specification occurs before this)

Question 6

What is the female pathway of germ cells after epigenetic reprogramming?

Answer 6

They quickly start meiosis. Almost as soon as the process is started, the cells go into mitotic arrest. The female cells at this stage are called oogonia, and are arranged in primordial follicles

Question 7

What is the male pathway of germ cells after epigenetic reprogramming?

Answer 7

The germ cells begin the process of meiosis - for longer than females. However they also go into mitotic arrest. The male cells are called spermatogonia at this stage and are arranged in primitive tubules called testis cords.

Question 8

Why is the potency of germ cells hotly contested?

Answer 8

Because germ cells only form one type of cell - gametes, suggesting that they are unipotent. But the gametes can form a totipotent zygote which induces pluripotent cells in the epiblast

Question 9

What are teratocarcinomas?

Answer 9

They are tumours which contain all 3 germ layer derivatives in them. Caused by germ cells mismigrating

Question 10

What are EC cells?

Answer 10

EC cells are cells derived from teratocarcinomas, and the first cells discovered with pluripotent properties.

Question 11

What are embryonic germ cells?

Answer 11

They are PGCs placed in culture, forming a pluripotent cell line - EG cells.

Question 12

Which important genes do PGCs express?

Answer 12

Many of the most important genes in the pre-implantation epiblast (and ES cells) including Oct4, Sox2, and Nanog

Question 13

What was the experiment that led to the discovery of imprinted genes and genomic imprinting?

Answer 13

Creating embryos with two maternal/paternal pronuclei which results in failed development. The DNA sequence was identical in all pronuclei so it must have been epigenetic information that differentiated the male/female pronucleus

Question 14

What is the epigenetic information that is present in developing embryos?

Answer 14

Different DNA methylation marks present on DNA inherited from the mother and father. These DNA methylation marks control gene expression in about 100/20000 genes that we have - this regulation is called genomic imprinting and the genes are imprinted genes

Question 15

What is the definition of an imprinted gene?

Answer 15

An imprinted gene is one that is monoallelically expressed in a parent of origin specific manner ie. expression depends on which parent it was inherited from, even if the gene is the same.

Question 16

How do males/females only pass on the male/female imprints if every cell in the body contains both male and female patterns?

Answer 16

When a PGC reaches the genital ridge, epigenetic reprogramming occurs where the majority of histone modifications and DNA methylation is erased. Then, a male/female pattern is established, as the cells go through either spermatogenesis or oogenesis. When the gametes combine, the maternal chromosome has maternal imprints, and paternal has male imprints

Question 17

What is the second wave of epigenetic reprogramming?

Answer 17

Post fertilisation. The DNA marks are stripped from the genome once more - but not the imprints. Also, 3 imprinted paternal genes survive the ovum attack on the sperm epigenetics

Question 18

What is the purpose of the second wave of epigenetic reprogramming?

Answer 18

Prepares the embryo to undergo development. Pre-implantation epiblast must be capable to make all cells of the body, so the epigenetic reprogramming gives an epigenetic blank slate.

Question 19

What is the state of methylation after implantation?

Answer 19

Cells rapidly accumulate DNA methylation as they move forward in development. While this DNA methylation is maintained in somatic cells, it is erased in the epigenetic reprogramming of PGCs

Question 20

Why is X-inactivation important?

Answer 20

Because if it didn’t occur the female would have too many genes because the X chromosome contains many more genes than the Y chromosome.

Question 21

What is X-inactivation?

Answer 21

The process that occurs shortly after implantation, where one of the two copies of the X chromosome is inactivated. The process is random in each cell. The inactivated X has its DNA heavily methylated and histones repressively modified. The inactive X chromosome is visualised as a Barr body

Question 22

What happens in PGCs regarding X inactivation?

Answer 22

In the earliest PGCs, there is an inactive X, like in all other cells at that time. However, during PGC development, the X is reactivated so that each egg has a normal X. This is completed around the time of epigenetic reprogramming

Question 23

How did John Gurdon prove that each somatic cell in the body retains the full complement of DNA?

Answer 23

Placed a somatic cell nucleus in an enucleated egg and obtained healthy frogs. This also provided evidence that somatic cells can be reprogrammed

Question 24

What does nuclear reprogramming mean?

Answer 24

A cell increases in potency, ie. the reverse of differentiation

Question 25

Why can an oocyte reprogram a somatic cell nucleus?

Answer 25

Probably related to the normal process of epigenetic reprogramming that occurs in the embryo. Also, the egg attacks the sperm and modifies its epigenome. It could be this mechanism that allows it to reprogram somatic nuclei.

Question 26

How can somatic nuclear transfer be used to benefit humans?

Answer 26

Could use somatic cell transfer to create an embryo, developing it to the blastocyst stage, and then harvesting human ES cells. These cells could then be used to make any cell type which could be transplanted into the patient

Question 27

Why can we not fuse a somatic cell with an ES cell to use for therapeutic cloning?

Answer 27

It was thought that we could fuse a somatic cell with an ES cell to avoid the need for somatic reprogramming with a human egg. The somatic cell does take on ES cell properties but will have double the genetic information

Question 28

What are the Yamanaka factors?

Answer 28

Oct4, Sox2, Klf4, and cMyc.

Question 29

What is the use of Yamanaka factors?

Answer 29

Introduced these genes into somatic cells and saw emergence of cells that look like mouse ES cells that could be grown and expanded. Can convert any adult somatic cell to a pluripotent state.

Question 30

What are induced pluripotent stem cells?

Answer 30

Somatic cells made pluripotent by the expression of Yamanaka factors - identical to ES cells.

Question 31

How else can somatic cells be reprogrammed?

Answer 31

Lineage switching, going back to a common branch point and differentiating again, or a direct conversion.

Question 32

What are the two different ways of setting aside the germline?

Answer 32

Preformation ie. a part of the egg is set aside for germ cells - there is always germ cells. Induction ie. cells get induced to become PGCs

Question 33

Why are PGC cells considered unipotent?

Answer 33

Because there is no evidence that PGC cells can incorporate into a blastocyst and create chimeras, and to keep the offspring to see if the cells enter the germline. They didn’t, so assumption is that PGCs can’t do it, so therefore not considered pluripotent even though they can make EG cells and teratocarcinomas

Question 34

Why is the ovum totipotent in some species but not others?

Answer 34

In some species, an unfertilised egg can be activated to form an embryo - parthenogenesis

Question 35

How does parthenogenesis occur?

Answer 35

Stress to the egg leads to refusion of the polar body, making the egg diploid again, or the egg never expels the polar body.

Question 36

What happens in mammals when parthenogenesis occurs?

Answer 36

Activated eggs can form blastocyst like structures than undergo a disordered version of development that results in teratocarcinomas

Question 37

How is epigenetics inherited?

Answer 37

Some TFs can stay bound to get the DNA back to its original state after mitosis - mitotic bookmarking. DNMT1A binds to hemimethylated DNA and symmetrically methylates it. Histone modifications not very clear.

Question 38

What prevents parthenogenesis in mammals?

Answer 38

Genomic imprinting. Same situation as embryo with two female pronuclei - two female pronuclei can be from two different cells or activated egg.

Question 39

How is genomic imprinting regulated?

Answer 39

Mainly by DNA methylation ie. methylated promoters = repression. Or, methylated upstream promoters but downstream unmethylated means antisense RNA which represses the gene.

Question 40

What is usually the function of imprinted genes?

Answer 40

Important behaviours such as maternal care, feeding, etc. And developmental processes

Question 41

Describe how Igf2 expression is regulated

Answer 41

Igf2 methylation before the gene determines looping so downstream enhancers activate the gene - occurs in paternal. Without this methylation mark, the loop is different and the enhancers don’t ensure expression

Question 42

Why is Igf2 expressed in males and not females?

Answer 42

Igf2 is a strong growth factor, males need to pass on their genes, but females need to preserve their resources, so they express a soluble receptor that soaks up Igf2

Question 43

What happens when Igf2 signalling is messed with?

Answer 43

No Igf2 -> Silver-Russel syndrome (maternal pattern) really small, failure to thrive. Beckwith-Wiedemann syndrome is paternal pattern, double dose of Igf2. Macroglossia, really big

Question 44

What is the purpose of epigenetic reprogramming?

Answer 44

In PGCs is to lay down appropriate imprints. To stop epigenetic mutations.