Cortical development and neural diversity Flashcards
Describe the methods of an experiment where they analysed hippocampal proliferation in-vivo
At P60, they did an I.P EdU injection and 24h later extracted the brain and collected coronal slices of the hippocampus.
They then stained the slices with EdU to stain the S phase (DNA replication) and Ki67 (growth factor). Ki67 stains during the cell cycle outside of S phase.
What are these stages after the S phase?
G2 (Protein synthesis and checks the duplicated chromosomes for errors)
=> M phase (Mitosis)
=> G0 phase (cell cycle arrest: Normal function/quiescence)
=> G1 (Cell growth: cellular contents excluding the chromosomes are dublicated)
What would EdU+, Ki67+ and EdU+Ki67+ NSCs indicate?
EdU+: In S-phase during EdU pulse, but not in cell cycle during sacrifice
Ki67+: NOT in S-phase during EdU pulse, but was in cell cycle during sacrifice
EdU+Ki67+: In S-phase during EdU pulse, and in cell cycle during sacrifice
What staining should also be done as a follow up?
Should do another immuno-staining to make sure they are stem cells. If you do this at different time points, the less EdU+ and Ki67+, as there are less stem cells and those which are still there are in deeper quiescence.
Name three complexes and checkpoints involved in the cell cycle and when they occur
The cyclin B-Cdc2 complex begins at the initiation of the M phase until the exit of the M phase. There is a checkpoint at the end of the G2 phase and at the exit from the M phase.
The cyclin D-CDK4/6 complex begins halfway through G1 until the initiation of the S phase. The cyclin E-CDK2 complex is activated from halfway through G1 until halfway through the S phase. There is a checkpoint at the initiation of the S phase.
What is the purpose of these checkpoints?
After S phase it is checked whether the DNA was replicated properly. During G1 there is a checkpoint for other intracellular components such as proteins to ensure they are split properly. If it is not sufficient there may be cell death or senescence.
What is the relationship of CDKs and cyclins?
Cyclins (CCNs) are activators of CDKs (Cyclin dependent kinases)
Describe the concentrations of 4 different cyclins over the cell cycle
Some are specific for a phase, others are globally important, there are many different cyclins:
The G1 cyclin forms a gaussian distribution over the entire cycle, beginning at G1, ending at M and peaking over the S phase.
The G1/S cyclin froms a narrow normal distribution spanning halfway through G1 and S phases, peaking halfway through.
The S cyclin slowly builds up from the end of the G1 phase, rising through the S phase and peaking in the G2 phase.
The M cyclin slowly builds up from the beginning of the S phase, rising through the G2 phase and peaking in the M phase.
Describe what happens when G1/S is present or not in the cell cycle
When G1/S is absent, CDK is inactive it does not influence its target genes and the S phase factors are inactive.
When G1/S is present it binds to CDK and activates it so it can phosphorylate targets and DNA replication enzymes are activated and the S phase begins.
What happens when M cyclin is present during the cell cycle?
When M cyclin is present it binds to CDK and activates it so it can phosphorylate targets. The spindle forms, chromosomes condense and nuclear membrane brakes down. The M phase begins.
What happens if DNA is damaged in G1?
p53 will interact with the CDK ihibitor P21 and this will bind to the CDK-Cyclin complex to inactivate it and therefore the cell is paused in G1. A cell will not enter S-phase when the DNA is damaged.
What therefore are the master regulators of the cell cycle and what relevance does this have?
Cyclin dependent kinase inhibitors are master regulators of cell cycle. They are chemicals used in cancer therapies (many in clinical trials)
Name three Cyclin-CDK inhibitors from one family and four from another
- Cip/Kip family: p21, p27 and p57
- INK4 family: p15, p16, p18 and p19
Describe a relevant point each about the members of the Cip/Kip family
- P21: induces DNA damage cell cycle arrest
- P27: upregulated in quiescent cell states
- P57: primarily involved in embryogenesis (generation of adult stem cells in the embryo)
What inhibitors inhibit what complex in the M phase?
p21, p27, p57 =| CyclinB-CDK1
What inhibitors inhibit what complex at the start of the G1 phase?
p15, p16, p18, p19 =| CyclinD-CDK6 / CyclinD-CDK4
What inhibitors inhibit what complex at the end of the G1 phase?
p21, p27, p57 =| CyclinE-CDK2
What inhibitors inhibit what complex in the S phase?
p21, p27, p57 =| CyclinA-CDK2
What inhibitors inhibit what complex in the G2 phase?
p21, p27, p57 =| CyclinA-CDK1
What happens if you knock out or upregulate p21
p21 is very important in maintenance, if you knock it out you lose your stem cells. If you upregulate it more stem cells go into quiescence.
How are CDK inhibitors upregulated?
CDK inhibitors are regulated at multiple levels:
* Transcription
* Phosphorylation (translocation)
* Binding partners
What are the Cip/Kip family of CDK inhibitors regulated by?
- P21 transcription is regulated by p53 (DNA damage)
- P27 transcription is regulated by FOXO3 (stress)
- P57 transcription is regulated by Notch/Hes1, BMPs (embryogenesis)
So CDK inhibitors are tumour supressors?
CDK inhibitors are not simply tumor suppressors, but are involved in an array of
processes, e.a. apoptosis, transcription and migration
How does cell cycle affect NSC fate decisions? (2)
Two main hypotheses on neural fate determination by cell cycle
cell cycle hypothesis
* G1 length defines the amount of cell fate determinants (e.g. Ascl1) and cell fate
Hypothesis of unequal inheritance of factors
* The distribution of cell fate determinants over two dividing cells defines cell fate