The germline and reprogramming

Question 1

What are primordial germ cells

Answer 1

First germ cells, Precursors of gametes which following meiosis develop as haploid sperm and eggs
unipotent but express many pluripotency genes and can form pluripotent tumours and stem cells

Question 2

Compare germ and soma

Answer 2

Those cells which enter the germ (or germline) will eventually form sperm or egg
When the egg or sperm go on to contribute to a new embryo then those two germ cells will live on in the next generation
Therefore, the germ forms the perpetual connection between generations and is essentially immortal.

In comparison, the soma (or somatic cells) are finite. Even those somatic tissues which are renewed with tissue stem cells will eventually die with the organism.

Question 3

What is the process where PGC are induced

Answer 3

PGC specification

1. PGCs go through the primitive streak and found at the back end of the embryo in the extraembryonic tissues
   
   2. PGCs then migrate into the developing gut and head northwards until the reach the developing gonads (genital ridges)
   3. They exit the hindgut and colonise the genital ridges
   4. This represents their final resting place and will continue to develop within the genital ridge
   5. Next event will be epigenetic reprogramming which differs in male and females

Question 4

How could you test if PGCs themselves are pluripotent

Answer 4

Ø Prevent PGCs to become EG cells
Ø Remove LIf, inject PGCs into blastocyst to check if it form organism
Ø If PGCs still grow without LIF in culture, and make various cells

Question 5

Describe epigenetic reprogramming

Answer 5

where germ cell development differs in males and females. SEx specification is by gonadal somatic cells and males turn on SRY and trigger downstream cascade while SRY is not expressed in females

Question 6

What occurs after epigenetic reprogramming

Answer 6

Females
• Start meiosis and paused and cells go into meiotic arrest
• Cells are called oogonia arranged in primordial follicles

Males
• Cells are called spermatogonia and arranged in primitive tubules called testis cords
• continue to proliferate for longer period of time but also pause during mitotic arrest

Question 7

Describe potential in Soma and Germ

Answer 7

In soma, loss of potency when cells specialize and pluripotency does not last long.

In germ, restricted potential as they can only make sperm or egg. They make zygote which is totipotent but after 4 days of development of embryo will include pluripotent cells again (pre-implantation epiblast)

Question 8

What happens when PGCs are in culture

Answer 8

they form pluripotent stem cell lines called embryonic germ cells that contribute to chimeras including germline when injected into blastocysts including the germ line

Question 9

Explain genome imprinting

Answer 9

• One that is expressed in a parent of origin specific manner
  
  • Genes whose expression is determined by the parent that contributed them
  • In the same nucleus of a cell, there can be two identical copies of a given gene but only one of these genes is expressed
  • DNA methylation markers are present in sperm and egg, retained in every cell of the developing embryo- including early PGCs

Question 10

Why do PGC undergo epigenetic reprogramming

Answer 10

to only pass male marks and female only passes female marks

Question 11

Describe epigenetic reprogramming

Answer 11

• Includes genome-wide erasure of majority of DNA methylation marks including imprints
  - Much of the extra epigenetic information is erased from DNA
  
  • Once Imprints erased it its possible to establish a male pattern a female pattern as germ cells go through spermatogenesis or oogenesis
  • Every sperm will carry male pattern of imprints and egg the female pattern
  • when these 2 gametes are combined, the maternal chromosome will carry female imprints while the paternal chromosome will carry male imprints so normal development can proceed.
  • these marks are erased early in germ cell development (prior to sex-specification) so that the correct, sex-specific DNA methylation marks can be laid down in sperm and egg during gametogenesis. Then the cycle can start afresh

Question 12

Why is it essential to erase imprints

Answer 12

to prepare PGC to enter meiosis , as germ cells accumulate imprints and DNA methylation when cells go through gametogenesis

Question 13

What occurs after fertilisation in epigenetic reprogramming

Answer 13

second wave epigentic reprogramming of demethylation to prepare embryo to undergo development and important for pluripotency
- After implantation-> cells rapidly accumulate DNA methylation
Will be erased once again during epigenetic reprogramming in PGCs and cycle continues

Question 14

Why is x-inactivation needed

Answer 14

• Female cells have 2 copies of X chromosome which contain many more genes than Y chromosome
  
  • if there was no mechanism to compensate for this, then female cells would get a double dose of a lot of genes

Question 15

Describe the process of x-inactivation

Answer 15

1. Pre-implantation epiblast both X chromosome are equivalent and active
   
   2. After implantation, one of these two copies becomes inactivated
   3. Process is random with one of the X chromosome shut down and marked with repressive histone modifications and heavily methylated DNA
      
      • Randomness of X-inactivation protects women from X-linked diseases

Question 16

what is the importance of x-inactivation

Answer 16

• this process is inherited prior to gastrulation- and if it is blocked gastrulation does not proceed normally
  
  • important for correct gene dosage to be present prior to the onset of gastrulation- a stage which complex cellular processes just be finely tuned
  • the random pattern of X-inactivation is what protects women from X-linked diseases- and the random pattern can be visualised in the coat of calico cats

Question 17

What happens in x-inactivation in PGCs

Answer 17

Earliest PGCs have one inactive X and is activated during PGC development so egg receives an equivalent chromosome that support development

Question 18

What is nuclear transfer

Answer 18

nuclear reprogramming that places somatic cell into enucleated egg and retains full complement of DNA producing evidence that somatic cells can be reprogrammed

Question 19

Why can oocyte reprogram somatic cell in this way

Answer 19

• After fertilisation, the egg ‘attacks’ the sperm and has to modify its whole epigenome
  
  • it is because the sperm nucleus arrives in a very odd state- packaged in protamines rather than histones for instance
  • it is possible that the normal mechanisms present in the egg to attack sperm, coupled with the normal epigenetic reprogramming that occurs in the pre-implantation embryo, is what allows this different sort of reprogramming to take place

Question 20

What are induced-pluripotent stem cells

Answer 20

• type of pluripotentstem cell that can be generated directly from a somatic cell

- Can be injected into the blastocyst and form chimerases and contribute to germline
- Identical properties to ES cells 
- Patient specific cells

Question 21

Describe the discovery of IPs

Answer 21

• Made a library of all TF that were known to be important in mouse ES cells
• Introduced these genes into somatic cells using a retroviral vector
• Some time in culture, the emergence of cells that looked like mouse ES cells
• Refined this method and only needed 4 TF - Oct4, Sox2, Klf4 and cMYc
- These TF is possible to convert any adult somatic cell to a pluripotent state