Neural Tube Regionalisation Flashcards
Marysias last half of lecs
What are the two fates of the neural stem cells in the neural plate?
Brain cells or spinal cord cells
Why are the cells in the axial mesoderm and their order important?
The neural plate which lies above each section of cells leads to the differentiation of either brain (above precaudal) or the spinal cord (above notocord)
How is the wnt pathway relevant in the differentiation of the axial mesoderm?
- Axial mesoderm under the neural tube
- Pre- caudal mesoderm expresses Wnt antagonists
- This means there is little to no Wnt signalling in the anterior portion, giving rise to the brain
- The notocord does not express Wnt antagonists so there is high Wnt signalling in the posterior portion, giving rise to the spinal cord
Give an example of a Wnt antagonist
Dkk (Dickkopf)
How do we know that the early neural tube is becoming regionalised?
- Looking at the expression of particular transcription factors (in-situ hybridisation) or looking at the binding if proteins in immunohistochemistry
- Sox2 going into a ‘keyhole’ shape, present throughout the whole structure as it is part of the dorsal mesoderm cells
- Otx2 is specific to the future forebrain
Give the molecular details about Wnt signalling, which genes ‘turn on’
When wnt is antagonised, Otx2 is ‘turned on’ to create forebrain cells
When wnt signalling is present, Hox cells are ‘turned on’ and spinal cord cells differentiated
How do we know that the Pre-caudal mesoderm is the source of signals that regionalise the neural plate to a brain identity?
- Neurula anterior mesoderm grafted into early gastrula induces an extra head with eyes and forebrain
How do we know that the ability of the PM to promote brain is mediated by Wnt antagonists?
- Over expression of Dickkopf leads to a much larger forebrain
- Dickkopf means fat head in german
- Knockout of Dickkopf means the forebrain is lost
What forms causing the neural tube to bend?
Flexures
What are responsible for the most posterior part of the nervous system?
Neural mesodermal progenitors
How does the spinal cord become regionalised into distinct domains?
- Different nerves will arise at different anterior and posterior levels of the spinal cord
Different domains defined through varied expression of Transcription Factors known as Hox genes
How is the expression of these Hox genes tested and why is it hard?
- Loss of Function studies (knockouts)
- Difficult due to there being so many Hox genes and a lot of them have the same expression patterns and do the same things
- Expensive
- Has been done in regions where there is less overlap such as the anterior regions
Give an example of the importance of 2 Hox genes in the formation of the hindbrain
Part of the hindbrain called r4 and r5 are characterised by the transcription factors Hoxa1 and Hoxb1, if these 2 genes are knocked out then these regions of the hindbrain never form
When the neural tube is being formed, where do the epidermal ectoderm cells go?
Surround the neural tube and fold over
What are neural plate border cells?
- A ‘third born cell’ from the ectoderm cells
- They develop into Neural crest cells
- These neural crest cells then go on to form the Peripheral nervous system
How are neural plate border cells formed?
- Due to a diffusion gradient, recieves an intermediate amount of BMP signalling which leads to its differentiation
- Neural stem cells recieve no signalling due to release of antagonists, epidermal receive lots of signalling, neural plate border cells receive a bit
What are neural plate border cells important for?
- Crucial for neural crest formation and hence peripheral nervous system development
- Roof plate formation and dorsal neural tube patterning/differentiation
- Final step in neurulation, closing the neural tube
Explain neural crest cell formation
- An early border (neural plate border cells) established at the interface of the induced neural plate and surface ectoderm
TF called Msx1 is produced- caused by intermediate level of BMP signalling - Msx1 and other genes form the neural plate border progenitor cells
- Other transcription factors are upregulated in neural plate border cells: c-Myc and Snail
These give cells stem cell like behaviours and they become multipotent, turning into neural crest cells - In response to c-myc etc. genes that control proliferation, multipotency and survival are transcriptionally activated
Neural crest cells delaminate from border region and begin to migrate away
What is an epithelial to mesenchymal cell transition?
- Cells lose their epithelial properties such as apical-basolateral polarity, tight junctions etc.
- Have more mesenchymal properties such as the ability to migrate
What do neural crest cells give rise to?
- Sympathetic
- Parasympathetic
- Melanocytes
- Adrenal medulla
- Shwann cells
What are the different cell types that neural crest cell give rise to determined by?
- Position of origin of neural crest cells determined by which Hox genes they express
- of generation of neural crest cells
- Migratory pathway and the signals they encounter en route
Some neural crest cells stay at the very dorsal part, why?
They help to ‘zip up’ the neural tube
Where are the roof plate cells located?
Dorsal midline of the neural tube
What do roof cells do?
- Begin to upregulate BMPs which then secrete locally into the un-patterned neural tube
- BMPs then induce expression of transcription factors called Pax genes
What do Pax genes do?
- They act intrinsically to cause neural tube progenitors to acquire ‘dorsal neural tube identities’
- Also induce BMPs, initiating a relay of BMP signalling cascade in dorsal neural tube cells, ‘cascade’
Why do you get different progenitors along the dorsal axis?
Different BMPs induce different cell types
What is the floor plate?
- A part of the neural tube directly (!!) above the notochord but in the midline so its below the roof plate
- Formed by specialisation from the axial mesoderm which lies beneath it (the notochord which will give rise to the spinalcord)
In what formation do neurons develop around the midline?
With bilateral symmetry
What does bilateral symmetry regarding neuron development imply?
The position of the notochord and the floor plate may indicate that they make secreted factors that direct the development of ventral neurons
How was the indication that the notochord may secrete factors which direct ventral neurons tested?
Gain of function:
- Notochord from donor is put in a host embryo at an ectopic position leading to a secondary floor plate and new pools of secondary motor neurons
How is Shh (Sonic hedgehog) relevant in the morphogen ventral neural tube gradient?
- Shh (sonic hedgehog) was found to be upregulated as the axial mesoderm forms - first expressed in the notochord and secretes Shh protein
- Shh protein detected beyond the floor plate cells, diffusing out of the notochord and the floor plate
- A gradient of the Shh protein can be detected in the ventral neural tube, Shh cannot be detected anymore around halfway up
- As a consequence you start to see cells that were initially all Sox2 positive now expressing other TFs.
What is meant by positional information?
Idea that a cell differentiates according to where it lies relative to neighbours that signal information
How is does Dorsal-ventral patterning include antagonising pathways?
- The BMP and Shh pathways antagonize each other
- BMP antagonists are still expressed in the organizer-derived axial mesoderm
- BMP signaling inhibits the expression of ventral genes, preventing the formation of ventral cell types.
- Shh signaling inhibits the expression of dorsal genes, preventing the formation of dorsal cell types.
- Together the opposing gradients of BMPs and Shh pattern the DV axis
Why do different types of neurons differentiate at the same D-V position along different parts of the A-P axis?
- Shh coming from ventral cell groups intersects with the earler-established A-P domain of the neural tube
- Cells respond by changing fate in accordance with their position in the grid
What do Shh and BMPs do at the early stage?
- Confer a Dorsal-Ventral pattern of transcription factors on progenitor cells
- These TFs are the upstream ‘master’ regulators of particular neuronal fate
Why don’t all progenitors become neurons?
- Some daughters stay as progenitors next to the lumen in a region called the ventricular zone
- The other daughter will differentiate into a neuron and will migrate sideways to the outer portion
What decides id the division of a progenitor is symmetrical or asymmetrical?
- Symmetric: Both daughter cells receive a bit of the membrane determinant
- Asymmetric: One duaghter will have the membrane determinant and one wont
What is interkinetic migration?
- Throughout the stages of mitosis the nucleus gets further away/closer from the lumen
- At cytokinesis the lateral attachment is lost, the cell divides and the lateral attachment reforms
How do you end up with differentiated sisters on the end/outer portion after migrating outwards?
- Neural tube gets wider so the neuroepithelial cells have to change their morphology and become radial glial cells
- Radial glia can divide asymmetrically, giving rise to one daughter which is like its mother – ie a radial glia (stem cell), and a 2nd daughter that will differentiate to a neuron.
- This daughter uses the scaffold provided by its sister to migrate away from the ventricular zone
What is the role of the Notch Pathway?
Used in asymmetric division of progenitor cells
How does the notch pathway work?
- The two cells are capable of producing Delta (an inhibitory signal) whilst having receptors for this signal (Notch)
- Bias is introduced so one cell starts making more Delta which means the second cell receives more Delta and the notch signalling pathway is activated
- This activation of the notch signalling pathway stops the transcriptional activation of acaete scute which is responsible for upregulating the gene that produces the inhibitory signal - this stabilising the change
- The first cell, receiving no delta and therefore no notch signalling means it produces more and more achaete scute
- Once the threshold level for achaete scute is reached, the cell differentiates into a neuron
What makes the first cell become a neuron?
Threshold level for the amount of achete scute produced in first cell
What do drosophila have instead of a neural tube?
- They have a neurogenic region which have activated ‘proneural genes’
- Only a few of the cells containing proneural genes will turn into neuroblasts which delaminate into neurons
How was the Notch pathway discovered in drosophila?
- By looking for drosophila mutants in by which no cells became neurons, ‘proneural mutants’ (e.g. mutant in Achaete scute)
- Or by looking at which drosophila mutants produced more neurons: neurogenic mutant more neurons are form e.g. if the cell does not see Notch
How can all of this be relevant for looking at congenital conditions? (from birth)
- Mice in which Shh is deleted show holoprosencephaly and cyclopia
- This means the mouse only has one central eye as there is no fully formed ventral midline and there is no bilateral ventricles forming being the brain is fused
- Shows anything interaction with the SHH pathway leads to this , applied in studying humans
How can we make purified neurons?
- Embryonic stem cells capable to giving rise to every single cell are treated with Wnts to get a posterior fate or Wnt and Shh to get ventral and dorsal progenitors
- These can be coaxed to become neurons through Notch signalling
- Due to the intersection of Shh with factors that govern A-P regionalisation, different types of neurons are born at the same level along the A-P axis
- Can make hypothalamic neurons and midbrain DA neurons for e.g. which are degenerate in Parkinson’s disease
How can we use this topics information in making Induced-pluripotent Stem cell neurons?
- Differentiated cells can be reprogrammed to go back to pluripotent stem cell fate
- A number of TFs can do this
- These IPS cells can be used in the lab and have factors added that control differentiation in the embryo
- Leads to dishes of purified populations of human neurons for example
- Good because there is no need for an embryo- much more ethical, easily accessible and individually specific
How can you generate human gastroloids from human IPS cells?
- Aggregates of either embryonic stem cells or IPS cells and with the right conditions, axial organisation of post implantation embryos can be recreated
- Gastroloids from some species can develop beating hearts
