Neurogenesis Flashcards
(21 cards)
What are the 4 stages of neurogenesis
Clonal restriction, precursor selection, neural identity and then path finding.
Describe how ectoderm cells become nerves in drosophila development.
Ventral ectoderm cells delaminate and differentiate into neuroblasts. These then undergo sucessive asymmetric divisions to form ganglion mother cells which can then divide and differentiate into nerve cells or glia.
For example in the fly PNS there are many sensory bristles, one unit involves 4 cells one of which is the nerve. These all come from a ganglion mother cell
Why is the development of the nervous system describe as having plasticity
If you kill one of the neuroblasts there is not phenotype as a result - the embryo can accommodate
What is an equivalence group?
cells that could all enter into one cell fate but don’t
Describe the seydoux and greenwal model for AC vs VU differentiation in C.elegans
2 cells in the C.elegans gondadal primordium (24aaa and 21ppp) can enter into two different cell fates - either an anchor cell or a ventral uterine cell. If you kill all other cells leaving either the 24aaa or 21ppp then either way they become the AC cell so this is default.
LOF mutation in the lin-12 gene results in both becoming AC and GOF result in both becoming VU. If one cell in Lin-12+ and the other is Lin-12- then everytime the lin-12+ becomes the VU.
Lin-12 is a receptor so the signalling cell becomes AC and the receiving cell becomes VU. Negative feedback stops receiving cell from signalling.
Describe how Notch function was characterised in Drosophila
LOF notch mutations result in neural hyperplasia and is lethal. Our equivilance group is the pro neural cluster, NB is default and ectoderm is the alternate fate. If we use mitotic recombination and so varying marker Notch doses we can show that at the border lower nothc doses always adopt the NB cell fate.
Notch is similar to Lin-12 containing a similar binding domain and this pathway is conserved in vertebrate neurogenesis. Evidence for receptor function - truncation of extracellular domain acts as if LOF.
Describe Delta function was characterised in drosophila
Delta LOF is also lethal and also results in neural hyperplasia . Repeat mitotic recombination experiment for delta. - Sensory bristles with higher dosage always adopt neural fate.
Delta has a DSL segment that can interact with Notch. Dual antibody staining of Notch and Delta show they overlap so are probably interacting.
How was E(spl) discovered - describe the function of this gene
Mutant screen in mutants. mutation in E(spl) gene enhanced the phenotype of a notch mutation called split.
E(spl) is a family of 7 genes all with HLH domains indicating it is a TF. these 7 genes have a functional overlap as you need to mutate many of these to get the phenotype.
Using GAL4 UAS can missexpress E(spl) and show that you lose sensory bristles - proved using fate map.
How can we determine whether E(spl) is influencing NB vs epidermal fate or A vs B fate in sensory bristle?
Using an SMC enhancer trap along with an over expression of E(spl). SMC cells are missing so must be preventing the formation of NB. WT function must promotes epidermal fate or antagonise neural or both
Are there any examples of proneural genes?
Achaete (ac) causes loss of microchaetes and reduced macrochaetes
Scute (sc) causes loss of macrochaestes and reduced microchaetes
Hairy (h) = extra microchaetes
Extra macrochaete = (emc)
WT function of ac and sc must promote NB function.
Describe the AS-C family of genes and their relation to daughterless
Larger family all with bHLH domains. Form dimers and affect transcription.
Da also has a bHLH domain
Gel retardation assays and gene reporter assays (done by transfecting e box domain (bHLH binding site) to a reporter gene) showed that Da can form heterodimers with sc and ac but ac and sc can’t form heterodimers or homodimers.
Who do sensory bristles only form in some places?
Da expression is ubiquitous
sc and ac is specific and corelates to where SOs form in the fate map.
Why doesn’t hsp-sc cause ubiquitous SO formation?
There is an antagonizing activity. emc and h both have opposite effect in mutant studies. emc has a HLH domain but no basic region.
Gel retardation assays and reporter gene assay show it forms heterodimers with sc and ac but that this inactivates the TF because there is no b region.
emc expression was origionally thoght to be ubiquitous but now shown to be reduced in the AC SC regions.
How do pro neural genes interact with neurogenic genes?
E(spl) alsp has a bHLH domain. When over expressed ac and sc function decreases.
E(spl) forms a homodimer and the last 4 aa attract groucho - a co repressor, what ever you put these last 4aa on it attracts groucho.
ac and sc both have e box domains in their promtors. E(spl) forms homodimers and binds to the e box. Then it attracts groucho causing repression of the neural fate.
How does DL interact with Notch to stimulate E(spl)?
Dl causes extracellular proteolytic cleavage event in Notch. This results in an intracellular cleavage event in notch. the protein produced (nintra) binds to a gene product called suppressor of hairless and together then stimulate E(spl) expression.
Why is control of proliferation important in neurogenesis and how is it achieved?
Neurons are produced progressively and the time that the neurons arrive is important for their fate. Controling proliferation is therefore very important to regulate this.
Dl achieves this by inhibiting differentiation in neighbors. When a new neuron is produced it transiently expresses Dl. This prevents any other neurons from differentiating and so creates a negative feedback loop controlling proliferation.
This was shown in chick eyes (easy to analyse and big). Over expression of DL caused a reduction in neural cells and under expression caused an increase in neural cells.
Why is neurogenesis slightly different in fly eyes compared to normal.
Normally proliferation occurs first and then through asymmetric division there is a gradual differentiation.
In the fly eye there are a pre prepared pool of cells. One the G8 cell differentiates and then recruits from this pool. Morphological furrow can be viewed. as a wave of differentiation across the eye.
How is NB cell lineage controlled?
With each asymmatric division the NB pushes a new GMC internally. Each one of these GMCs forms a different cell type and has a different TF profile.
This profile is determined by the TF profile that the NB had at the time of its birth. If the TF in a specific profile is under expressed of KO then that cell is skipped, dies or takes on the profile of a later GMC. If it is over expressed then all GMCs past that point become that fate.
However the TF profile does not decide cell fate it only relates to birth order of the cell. There are two patterns of TF, one is V-D the other is A-P. This creates a code of different profiles which decide ultimate cell fate. On top of this are cells secreting growth hormones that secrete out to different concentrations and intersect with the individual TF profile from a cell to indicate the cell type.
What is the competence window
A NB lineage can be forced to express a TF even after it normally would but only up until a point. After this window is closed it can no longer express tat TF.
This is because the locus for that TF slowly associates with the nuclear lamina over time.
How was the filopodia disocovered?
Some cells are programmed to enter into the epidermal fate even thought they aren’t in contact with a NB delta producing cell - so why don’t extra NB form.
Delta does have a secreted form but it is not active. Could it be transported across cells?
Use Hsp:FLP and tub:>STOP>GAL4, UAS-CD8;GFP
CD8 is a membrane tag. This showed that 30% of cells have something called a filopodia - extension of the cell membrane.
How do we know filopodia are used to signal with delta and notch?
Using a Dl-GFP fusion and express under UAS with a neuralised:GAL4. Cross to filopodia markering strain and can see that Dl is present on filopodia.
Decreasing Dl function decreases the amount of filopodias and their length.
Monitor Notch function with E(spl) LacZ and increase Dl function - results in increased Notch stimulation. Can also block filopodua formation using the Ezrin DN truncation.
Blocking filopodia formation reduces Notch singalling.
Finally - normally microchaete emerges in waves to fill gaps. If you stop filopodia formation then there is an increase in microchaete density because Notch stimulation is decreased.
