Lecture 2 Development

Why is it important to understand the relationship between info in the nucleus and regularoy moelcuels in the coplasm

• key to understanding cell differentiation and its control

• exposing differentiated cell nuclues into cytoplasm of another was able to change its pattern of gene expression

Cell fusion experiments

1. Chick red blood cells with human tumour cells

Experiments where nucli exposed to forign cell cytoplasms Experiment 1

E.g Chick red blood cells fused into human cancer cell

→ red blood cell nucleus enlarges

• chicke specific proteins begin to be synthesised in the fused cell

THEREFORE: There are clearly factors transferred through the cytoplasm, have reverse in the inactivity of the nuclues

• transciption of the chicken genome is reinitiated

conclusion: Red blood cell nucleus takes a protein from the tumour cell cytoplasm and resumes transciption!

Experiemnt 2

• Aminocyte→ undifferentiated cell i think?

• Put into the cytoplasm of a mouse muscle

• the human aminiocyte now extresess its own muscle stuff

• So has switched on the muscle genes in the huamn from stuff in the mouse cytoplasm

Experiment 2

Human liver cell fused with a rat muscle cell (large multinucleate cell)

Result:

• Expression of human liver-specific genes turned off

• transciption of human muscle-specific genes switched on

→ expression of both human and rat muscle proteins can be detected in the fused cell

• bascially made the liver cell now express stuff as it it were a muscle cell

• Due to stuff in the ctyoplasm of the mouse cell

These experiments therefore show

• gene expression is regulated by factors present in the cytoplasm of differentiated cell

What are these factors? Experiment 1 method

1. Transfecting cells with cDNAs made from mRNA taken from proliferating myoblasts

why?: some of these muscle specific mRNAs would encode regulatory proteins controlling muscle cell differentiation!

Experiment 1 Results

• A single cDNA for a genes (MyoD myogenic differentiation gene)

• transfected into differentiated fibroblasts

• causes cells to switch fate→ MUSCLE CELLS

What does MyoD encode for

• DNA binding protein transciption factor

  • binds to the upstream sequences of muscle specific genes

  • activates them

also

• Activates its own transicption

THEREFORE: once started

• MyoD preotin ensures that a stable feed back loop operates

• maintain the expression of muscle specific genes!

Further information we know about this network with MyoD

• combined effect of these factors and other gesn downstream

• Creates multiple feedback

The prescence of multiple feedbacks means

• once differentiation is established

• it is very stable!

But how do such stabilising loops are initiated as the embryo develops?

• e.g for MyoD→ a signal leads to the onset of muscle differentiation

To answer this question need to understand…

• how the realtion between nucleus and cytoplasm begins

The beginnings of embryonic development

1. Prior to fertilisation, mature egg is developmentally paused

2. fertilisatieon releases the pause→ rapid cell division and DNA replication cleavage

What does celavage result in

• each replicated set of chromosomes ecoming associated witha different fraction of the original egg cell cytoplasm

(different processes in Xenopus and Drosophila but the same outcome at hthe end)

In the Xenopus

• egg is simply divided into a ball of cells

In Drosophila

1. first replicated without cell division→ form a syncutium

2. migrate to the surface of the egg

3. where each nucleus becomes surrounded by a cell membrane

How is this cleavage process programmed into unfertilised egg cell

• in maternal gene prouducts (mRNAs and proteins)

• put into the egg by the mother

→ sufficient to carry the embryo through the early runds of cell division and nucluear replication

What is the Mid-Blastula Transition MBT

• the event when the embryo begins to take charge of its own development

• Trasncitpton of the embryonic genomes starts

and also

• Cell cycle lengthens

  • To include a growth phase

  • rather than repeated cycles of replication

When does this happen in Xenopus?

• Until the cell cycle 12

similar event also in Drosophila

Showing unequal distribution of proteins and RNAs in the egg cell

• using antibodies and in situ hybridisation

→ show they are partitioned by cleavage

e.g→ Vg1 transcitps are localised to the vegetal pole of the frog egg→ bright fluoresecence

Segregation of protein granules in nematode C.elegans

• into germ cells→ future gametes

How does this happen?

• Asymmetric distribution of the granules in the egg cell

• before it begins to divide

Does the unequal distribution of proteins and RNAs in the egg cytoplasm cause a crticial determinat of fate of the cells generated as this cytoplasm is subdivided by cleavage?

• Drosophila provides a simple test for this

What the drosophila show

• the germ cells in the fly are produced by cells

• at the posterior pole of the embryo

→ IS there something special about this part of the egg cytoplasm→ that dictates the formation of germ cells, rather than any ther cell type

What is unique about what these pole cells include in them

• the cytoplasm of the most posterior part of the egg

Experiment with pole cells

1. Posterior cytoplasm is transplanted to an anterior position in another egg

2. Nuclei at this position normally contribute to head strucuture

3. BUT→ as the embryo cellularises…

4. These cells include posterior egg cytoplasm

→ Do they now mark germ cells rather than head strucutures?

Experiment continued

Yes they do:

• clearly shows that there is something in the posterior egg cytosplasm

• That dictates germ cell production

• When it is included in formaing cells

This germ cell deteminant is known to be

• theproduct of the gene oskar

Questions to be asked next

• how we can find out what is put into the forming egg cell by the maternal genome

• how we can identify these factors molecularly