15. cacner and developmental biology Flashcards
how many neurons are in the adult brain?
80 billion
give three ways that neurons can differ from one another
- neurotransmitters
- morphology
- receptors
when a stem cells divides what can it become?
another stem cell
a daughter cells - with less potential
why is very important to have self-renewal and differentiation balanced?
- too much self renewal and not enough differentiation - large tissue, hyperplasia, tumour formation
- too much differentiation and not enough self-renewal - reduced nervous system, small head and impaired tissue maintenance
when do NSC originate in Drosophila development?
in the embryo
what happens 4 or 5 hours in Drosophila development?
there is delamination of NSC from epithelial layer around embryo
>NSC undergo asymmetric division
when NSC undergo asymmetric division - what do they become?
another stem cells and a GMC (ganglion mother cell)
what do GMC tend to do?
divide once more to produce either neurones or glia
what from the epithelial layer of cells is translated into the epithelial layer?
the polarity - proteins at the apical cortex and proteins a the basal cortex
why is there asymmetric division
due to the components inherited by cells and the size of the cell
which cells is smaller: the NSC or the GMC?
the GMC
out of the apical and basal factors, which influence self-renewal and which influence differentiation?
> apical factors are important in maintaining self-renewal
>basal factors influence differentiation
what is asymmetrically segregated into GMC?
prospero - transcription factor that is held in the basal cortex of neural stem cells
what NSC divides and GMC is produced, what happens?
prospero is released from cortex and enters the nucleus
what is prospero key in?
key factor in switching the NSC from a self-renewing state to a differentiation state
what does CHIP stand for and what does it identify?
chromatin immuno-precipitation
>find where TF bind in genome
how does CHIP work?
cross link DNA with TF and pull down with antibody
>use microarray/next gen seq to identify sequence
what technique can be used to map protein-DNA interaction in vivo? and give a benefit of this
DamID - DNA adenine methyltransferase Identification
>does not require antibodies
how does DamID work?
> protein of interest fused to E coli enzyme that methylates adenines at specific sites
express at low level
where protein binds will methylate adenine
v little methyl adenine in DNA to interfere with results
cut out methyl adenine using enzymes that cut these locations
amplify and determine sequence
what sort of genes does prospero bind? and how was this determined?
it binds and represses cell cycle genes and NSC genes and activates differentiation genes
>DamID and comparing expression level with prospero mutants
where does prospero binds?
strongly to the intergenic regions of cyclin E genes
In terms of cancer, what is prospero?
a tumour suppresser
what is seen in prospero mutants and what can this lead to?
continue to self-renew and do not differentiate - keep dividing with no queues to differentiate
>this can lead to tumours
why are MARCM (mosaic analysis with a repressible cell marker) models useful?
you can make clones of cells during development that are mutants and label them with GFP, if entire embryo was mutant then it would die as embryo
prospero MARCM showed tumour formation in mice brains, these cells are dividing but are they really cancerous?
take cells and put them into abdomen of other fly - these tumours undergo metastasis
>tumours can be serial transplanted for at least two years
describe the protocol used to determine what was working with prospero to repress NSC genes and promote differentiation genes
> extract sequence from prospero binding regions
filter non-exonic regions and conserved sequences
top hit = palindrome, that looks nothing like prospero binding sequence i.e. suggests another factor binds near prospero
once the sequence for the protein that binds near prospero and help mediate differentiation was identified, what was done next?
yeast one-hybrid screen
>several copies of motif placed upstream of reporter gene
>transform cDNA libraries into yeast (all protein in embryo fused to AD)
>yeast that have factor which binds motif express reporter
what factor was determine to bind this conserved motif? and where is this expressed?
lola
>in the developing nervous system
what type of protein is lola?
BTB zinc finger TF
how many splicing isoforms are these of lola? and what do they all have
25
BTB dimerization domain
where is lola expressed?
post mitotic neurones - this is turned on as soon as the neuron is born
name the protein that is a marker for NSC and holds prospero at the cortex?
mira
what type of cells is lola expressed in?
neurons, not glia
what does lola bind?
lola binds and represses neural stem cell genes
>represses key NSC genes like deadpan
how many genes overlap between prospero and lola?
259
is lola a TS?
yes
>KO tumour cells formed expressed NSC markers and actively divided - normally undetectable levels of division in adult brain
what happens in terms of differentiation with lola mutants?
cells get signal to differentiate, but without lola NSC are not kept off and cells revert to NSC - gradually express NSC markers
>this forms tumours in the brain
what does the removal of lola result in?
de-differentiation in immature neurons
how does a neurone become mature?
send out axons and form synapses with other cells
what happens when lola is removed from mature neurones?
nothing
how will understanding what maintains repression of NSC genes in mature neurones affect therapeutics?
> better understanding of how some brain tumours form
better understanding of certain resistance to therapies
better understanding = better therapy
if we can control de-differentiation we can instruct brain to repair damaged tissue
how can we identify factors maintaining differentiation in mature neurones?
yeast 2 hybrid
>take lola bound to GAL4, this binds UAS of his3
>when bound to binding partner bound to AD there is expression of His3
how many colonies were identified in Y2H?
111 with good confidence
what sorts of things did the Y2H screen identify?
chromatin remodellers, TFs and lncRNA
what % of the hits in Y2H were lncRNA?
60%
what does lola recruit? and what is this key for?
NurD complex which moves around nucleosomes and recruits HDAC - promoting turn off of NSC genes
>this is what keeps neural stem cells repressed
why is lola no longer required in mature neurones?
the DNA is in its repressed state and so cells wont de-differentiate
name the lncRNA that is segregated into the GMC? and what does this express?
copa
>micropeptide of about 30 amino acids that interacts with lola and affects its function
what happens when copa is KO?
this results in fewer GMC dividing (normally divide once more)
what is the current hypothesis regarding copa?
> micropeptide binds and inhibits lola (cleave/degrade)
this prevents pre-mature differentiation
if lola acts prematurely then GMC stop dividing an will have one less neurone in brain
in terms of cancer what type of protein is this micropeptide?
an oncogene
describe the balance between symmetric and asymmetric division in the developing NS
NSC divide symmetrically amplifying the pool of NSC - occurs early in neurogenesis
>asymmetrically divides - maintain NSC whilst producing progeny.
what two types of signalling promote symmetric NSC division?
Notch and Hippo
what does activation of the hippo pathway lead to?
repression of transcription factor yorkie
what does over activation of the hippo pathway cause?
over proliferation of NSC
>results in hyperplasia (this is often the initial stage of developing cancer)
what happens when hippo is KO?
this reduces number of NSC
what is associated with brain tumours in humans?
activation of hippo signalling - promotes symmetric division to drive tumour growth
>it may be amplified and over expressed
describe notch signalling
delta ligand interacts with notch
>intracellular notch released into cell
>this enters nucleus and interacts with Rbpj
>this interactions promotes gene expression
what genes does notch signalling activate?
genes associated with NSC proliferation in the NS
what happens when notch signalling is inhibited?
neural stem cells asymmetrically divide into neuroblasts.
what happens when notch signalling is increased in drosophila? and how might this increased signalling come about?
this results in brain tumours
>increased expression of intra cellular notch
what happens when Rbpj is KO in mice brains?
there are fewer NSC symmetric divisions - Rbpj is required to drive normal proliferation
what is increased notch signalling associated with in humans?
brain cancer
in most human cases Notch promotes tumour growth, what does it do in other cases?
supresses tumour growth - context is important
what sort of inhibitors have are in clinical trials in terms of notch signalling? and what is bad about this?
gamma secretase inhibitors - this means intracellular notch cannot be cleaved
>not v specific and so there are lots of off target effects
why does inhibiting gamma secretase have lots of off target effects?
> notch is used in many other pathways
>this enzyme is not just used to cleave notch
