BMS381 Developmental Neurobiology Flashcards
What is the purpose of the central nervous system?
The Central Nervous System allows us to receive information from our environment both externally (from the peripheral nervous system) and internally (e.g. release of stress hormone)
How did nervous systems first arise?
- Nervous systems arose with multicellularity, allowing a variety of neurons to give flexibility and coordination
- Early neural cells arose from the surface layers – ectoderm differentiates into skin or neural tissue
What is the neuroepithelium?
Neuroepithelium is a one cell thick sheet of neural tissue. It is already fated to be neural meaning it can give rise to a neurone or a glial cell
What is the neural tube?
A single layered neuroepithelium, induced by the ectoderm on the dorsal side of the embryo
This then grows and elongates along the AP axis and rolls into the neural tube
Simple cell movements that underlie these morphological changes
What factors can govern gene expression?
Gene expression in a cell can be governed by extrinsic factors (morphogens) and intrinsic factors (transcription factors)
How does ectoderm acquire a neural fate?
Under the influence of transcription factors such as Gsc, the organiser expresses unique secreted products that are all antagonists of the BMP signal (chordin, noggin)
This causes the phosphorylation of SMAD157 and therefore the up regulation of transcription factors such as SoxD
This induces a neural fate in those cells
How does ectoderm acquire an epidermal fate?
BMP is not inhibited meaning SMAD157 is not phosphorylated
Indues transcription factors such as Msx1 and GATA1
Causes epidermal differentiation
What occurs in gastrulation?
As soon as the organiser induces the neural plate, the organiser self-differentiates into the axial mesoderm. The axial mesoderm then involutes and undergoes convergent extension
How does gastrulation lead to the formation of the AP axis?
- The first cells to migrate are the anterior endoderm cells, followed by the prechordal mesoderm cells. Thy migrate up and along These mark the future anterior of the embryo and will be important in building the head of the embryo
- The last cells to migrate are the notochord. These will underlie most of the body with the back end forming the posterior end of the embryo
How does gastrulation lead to the formation of the DV axis?
The axial mesoderm now underlies prospective neural tissue which is a dorsal structure. This is why the organiser is initially referred to as marking the future dorsal axis as well as the anterior/posterior axis
What does impression mean?
Individual cells leave an epithelial sheet and become freely migrating mesenchymal cells
What is epiboly?
A sheet of cells spreads by thinning
What is intercalation?
Rows of cells move between one another, creating an array of cells that is longer but thinne
How do we know that the early induced neural plate is anterior in character?
The organiser self-differentiates and undergoes convergent extension. If we experimentally stop development at this point in time, and look with molecular markers, we find that the neural plate is expressing markers that are later confined to the forebrain (telencephalon and diencephalon)
How does the neural tube develop its anterior posterior identity?-
- BMP antagonists and Wnt antagonists are maintained anteriorly in the prechordal mesoderm
- FGF, Wnts, RA are expressed posteriorly by the late organiser/ Node, promote growth and posteriorised
- Formed by establishing a regional pattern by placing two antagonistic molecules at each end of a forming (growing) structure
What does the neural crest give rise too?
The entire PNS
How are neural crest cells formed?
- BMPs are expressed by surface ectoderm cells, next to edges of induced neural plate
- Neural plate border cells are established by intermediate levels of BMP signalling triggering transcription factors (msx). These either develop into neural crest cells or are retained at the border to form roof plate cells
- BMPs, working with Wnt and Fgf signaling, initiate a cascade of events that will give rise to highly-potent, proliferative Neural crest cells
How does dorsalisation of the neural tube occur?
- Neural crest cells and roof plate cells are induced by the neural plate boarder cells
- Roof plate cells then upregulate BMPs
- Secreted BMPS diffuse into the dorsal neural tube. They induce expression of a transcription factors (Pax6, Pax7, Pax3, Lim1) that cause cells to acquire ‘dorsal identities’ and form dorsal progenitors
How does ventralisation of the neural tube occur?
- The axial mesoderm (notochord) start to produce Shh which acts as a morphogen
- This induces the floor plate which then also starts to secrete Shh – positive feedback.
- The Shh diffuses out of the floor plate and notochord and into the neural tube
- Induces the transcription factor Gli1 turning on expression of other floor plate genes – e.g. Shh itself
- Genes encoding different transcription factors begin to be transcribed and translated in different cells along the D-V axis
How can the role of Shh as a morphogen be investigated?
Immunohistochemistry
What are homeodomain transcription factors?
Master regulatory transcription factors
What was the purpose of the paper ‘Identification of a neural crest stem cell niche by spatial genomic analysis ‘ in 2017?
- The neural crest is an embryonic population of multipotent stem cells that form numerous defining features of vertebrates
- Due to lack of reliable techniques to perform transcriptional profiling in intact tissues, it remains controversial whether the neural crest is a heterogeneous or homogeneous (single stem cell) population
- This paper describes a novel technique that combines in situ hybridization with machine learning to examine complex gene expression in cells in the developing neural tube at single cell resolution
How did the paper ‘Identification of a neural crest stem cell niche by spatial genomic analysis’ in 2017 allow advancements in the field?
They were able to examine the expression of 35 genes at a time. Before this could only happen with two genes. This is much cheaper and efficient. Advances in microscopy allowed better resolution and the ability to visualise individual transcripts as a single dot
What is hierarchically clustering?
When a computer is fed large data sets and the computer sorts the data and categorises into the group
What did the paper ‘Identification of a neural crest stem cell niche by spatial genomic analysis ‘ find?
Used hierarchically clustering.
- The clustering revealed five distinct groups within the chicks dorsal neural tube.
- They pooled data together from three embryos and discovered two main cell populations
- Cells that express both pluripotency and differentiation markers and cells without a pluripotent signature
- These can be further clustered into different subpopulations of neural or neural crest cells. They express different stem cell markers
How is the majority of the CNS structured?
In columns and layers
Why is the majority of the CNS structured in layers ad columns?
- The development of the AP and DV axis
- Spatial morphogens on opposite sides of the spinal cord (eg. BMP, Shh) set up opposing concentration gradients causing differentiating cells to move laterally and form layers
- Time - Neurogenesis occurs in waves forming different layers per wave
What part of the CNS does not conform the column and layer structure?
The hypothalamus
How are the neurones of the hypothalamus structured?
Neurones are found in clusters called nuclei that are arranged in a patchwork manner
What is the function of layers in the CNS?
Allow organisation of function
Is there any organisation of function in the hypothalamus?
In each nucleus, there are clusters of neurones which can have completely opposite functions
What are the functions of the hypothalamus?
Controls the core body functions
- temperature, growth, electrolyte balance, metabolism, reproduction, sleep, stress (cortisol)
Maintains mental and behavioural homeostasis
- Desires, Mood, Anxiety, Sexual drives, Motivation, Trust, Aggression, Stress
What was first discovered about the development of the hypothalamus?
Shh loss of function studies (1996)
Shh knockout lead to dismorphology of the forebrain, cyclopia and holoprosencephaly
Lead to the understanding that Shh was important in the development of the ventral part of the brain including the hypothalamus
What part of the axial mesoderm is thought to be involved in hypothalamus development?
This focussed attention on the prechordal mesoderm, since this part of the axial mesoderm expresses Shh and underlies the anterior most part of the neural tube
The prechordal mesoderm doesn’t extend all the way to the front of the neural tube
A number of experiments showed that Shh deriving from the prechordal mesoderm induces a fan shaped set of cells in the neural plate immediately above it that themselves express Shh. What experiments could be used to show this?
Surgically ablate the pm: expect to see no shh induced
Graft extra pm: expect to see ectopic shh
Combine a piece of pm with a piece of naïve neural plate: shh induced
Same as 3 but pre-treat pm to block shh: should prevent effect
Conditional knock-down of shh in pm in vivo
What did the finding that the prechordal mesoderm induces a fan liked group of cells that express Shh focus attention on and why?
A part of the ventral midline of the forebrain on a ‘floor plate’ like structure
- The fan shaped Shh expressing cells stop abruptly at the end of the floor plate like structure (yellow)
How did the discovery of the fan like group of Shh secreting cells provide a theory of the unusual arrangement of nuclei in the hypothalamus?
- Shh will diffuse from the Shh producing cells. The midline cells secreting Shh will pattern across the AP axis (normal layer and column organisation) but the fan shaped set of cells at the end of the Shh secreting cells will cause Shh to diffuse in a circular pattern around the fan, patterned the DV axis as well as the AP
- Could the pattern of hypothalamic development be explained by the formation of arcs of progenitor territories, around the end of this floor plate.
Was the theory of - the fan like cells secreting Shh in an arc pattern being responsible for the interesting hypothalamic arrangement - correct?
- For a very brief period (0.5 days in chick), you do see these patterns. But they become very rapidly obscured
- Analysis of known progenitor markers at the end of the floor plate showed chaotic clustering around the edge of the ‘floor plate-basal plate’ territory.
- These patterns could not be explained by a simple Shh morphogen gradient or arcs of progenitors
How does the gene expression in the prechordal mesoderm change during embryogenesis?
- Expresses Shh - induces the set of fan-shaped ‘neural-plate’ like cells above it
- Expresses BMP antagonist (chordin, noggin, follistatin) whilst it expresses Shh to maintain anterior identity.
- It then loses the BMP antagonists and then upregulates BMPs.
What does the expression go BMPs in the prechordal mesoderm lead to?
- BMPs diffuse into the fan-shaped ‘neural-plate’ like cells and induce transcription factors called Tbx2, BMP2 and BMP7, Wnts, and Fgf10.
- They also upregulate all of the components which allow response to these signalling factors.
- Very rare to see this combination of factors (other place is neural crest cells)- induces a population of cells which will have stem cell properties.
What was the hypothesis for the experiment completed by Fu et al in 2017?
The prechordal mesoderm induces a hypothalamic stem cell that co expresses Shh, BMP4 and Fgf10
How did Fu et al (2017) test their hypothesis?
- They tested this by performing fate mapping studies to ask if cells secreting Shh, FGF10 and BMP7 give rise to many other hypothalamic cells and whether they self-renew (are they stem cells?)
- If you inject floor plate cells with a dye, it stays there – indicates that they don’t give rise to new cells/migrate and proliferate.
- If you inject same amount of dye into fan-shaped neural plate like cells, then you see it diffuse quickly into many areas of the hypothalamus-gives rise to many cell types and so is multipotent
What did Fu et al (2017)s experiment find?
Found that the hypothalamus derives from the Shh, BMP7 and FgF10 secreting cells that lie above the prechordal mesoderm
- As if you inject dye into fan-shaped neural plate like cells, then you see that these cells give rise to a large amount of the hypothalamus
Why did Fu et al (2017) think that the Shh, BMP7 and FgF10 secreting cells that lie above the prechordal mesoderm are stem cells?
Because this combination of signals is similar to neural crest (although NC doesn’t have Shh)
Why was the first experiment that Fu et al (2017) completed not sufficient to prove the cells were stem cells?
The experiment shows that the cells can differentiate and give rise to multiple cell types but it doesn’t show that the cells are self renewing
How did Fu et al (2017) show that the Shh, BMP7 and FgF10 secreting cells were self renewing?
- They injected red and green dye into the progenitor cells.
- There was always an area that appeared yellow as some of the descendants of the green cells and red cells remained in the domain – this domain continues to express Shh and FGF10
- This suggests that they are self-renewing and therefore a stem cell
What did fate mapping and marker analysis show about how stem cell differentiate and self renew?
- Cells grow out of the progenitor domain in one direction and other cells grow out in the opposite direction. While this happens, the central population is maintained as stem cells
What is meant by there being a proliferation front at the edge of each cell type domain?
There seems to be a proliferation front at the edge of each domain- therefore the highest amounts of proliferation occurs in cells that are downregulating Fgf10 and upregulating Shh
How is thought that stem cells differentiate into two different daughter cells?
- Stem like cell divides to give two daughters which look completely different – one retains Shh and other Fgf10 and BMP
- Shh is then secreted from that daughter cell to act on the other daughter cell to reregulate Shh
- This causes the daughter cell to become a stem cell again (self renews).
What is believed about the daughter cell of a stem cell that expresses FGF10 and BMP before becoming stem like again?
It is believed that the daughter cell that expresses FGF10 and BMP is a stem cell in a state of flux while it differentiates.
This is before the other daughter cells reinduces Shh so that it becomes identical to the mother cell (stem cell).
Why is there a population of Fgf10+Shh+ cells maintained throughout the life of the hypothalamus?
There are waves of neurons born in the hypothalamus throughout life from these progenitors
Explain how the anterior pituitary gland is formed
- The prechordal mesoderm induces the FgF10+Shh+ cell population and it moves away.
- In its place, there is an ingrowth of oral ectoderm which develops in and under the FgF10+ expressing cells, developing a pouch like structure.
- This Fgf10 and Shh signals to the ectoderm pouch cells telling them to upregulate a transcription factor called Lhx3.
- An ectoderm cell that expresses Lhx3 becomes Rathke’s pouch
- This will self-differentiate into the anterior pituitary gland
Explain how the posterior pituitary gland is formed
Some of the other FGF10+ cells give rise to a ventral out pocketing called the infundibulum. The distal cells of this out pocketing will self-differentiates into the posterior pituitary (diagram D) and the proximal cells will give rise to the median eminence
How is the median eminence formed?
The proximal cells of the infundibulum give rise to the median eminence
Give evidence for the involvement of Shh in the formation of the pituitary glands
Fu et al, 2017
- Wild type mouse and one treated with cyclopamine (disrupt Shh signalling)
- One treated with cyclopamine, Lhx3 isn’t there so Rathke’s pouch doesn’t form, infundibulum doesn’t form
- Results in a non-viable embryo
Why are the two pituitary glands often talked about together?
Both pituitary glands share the ability to release hormones but the hormones are released in a very different way
What is different about the way the anterior and posterior pituitary glands release hormones?
The posterior releases hormones made in neurosecretory neurones directly into the blood stream but the anterior secretes the hormones in response to a hormone releasing hormone from the hypothalamus
Give examples of hormones secreted from the posterior pituitary gland
Vasopressin and oxytocin (trust hormone)
How do axons from the hypothalamus reach target cells?
FgF10 guides them
How are hormones released from the posterior pituitary gland?
Neurosecretory neurones located in paraventricular nucleus produce vasopressin and oxytocin directly and are then transported to the posterior pituitary. Vasopressin and oxytocin (trust hormone) are then released into the blood supply of the posterior pituitary.
How are hormones released from the anterior pituitary gland?
- Another set of hypothalamic neurons project to median eminence
- They are neurosecretory and release hormone releasing neurohormones which are located in the arcuate nucleus and project to hypophyseal portal system in anterior pituitary
- These hormones are released into the portal capillary supply and travel to the anterior pituitary gland where they govern the release of hormones from endocrine cells
How do the difference cell types in the anterior pituitary control which hormone is released?
There are six different cell types make up the anterior pituitary. Each cell type is under the control of a specific neurone in the hypothalamus. E.g. Growth hormone is released from anterior pituitary when it detected growth hormone releasing hormone from the hypothalamus
What are tanycytes?
Radial glia in the hypothalamus
How are tanycytes formed?
At the proximal part of the infundibulum there are FGF10+ cells maintained as radial glia called tanycytes into adulthood
What is the role of tanycytes?
Some of these line the median eminence and are involved in acute homeostasis and others are FgF responsive multipotent and neurogenic stem and progenitor cells.
Give an example of how tanycytes can be used in acute homeostasis
- The release of growth hormone is controlled by growth hormone releasing hormone which is made by hypothalamic neurones throughout the day.
- These neurones project to the median eminence where the tanycytes block its release into the portal capillary.
- At night, the tanycytes retract and growth hormone releasing hormone can enter the anterior pituitary and growth hormone can be released. Therefore, only grow at night.
What first made us expect there was a stem cell in the hypothalamus?
The hypothalamus must be able to adapt to anticipate and meet new changing conditions (e.g. puberty, growth, pregnancy, hibernation)
- Key points in life need to generate new neurones to meet its needs
How was the distribution of tanycytes investigated?
Mouse model organsim
- dissect brain
How are tanycytes arranged in the hypothalamus?
- The hypothalamus is arranged around the third ventricle of the brain and tanycytes extend around the third ventricle forming the region
- They have long basal extensions.
- Some tanycytes project into the median eminence itself and others project to particular nuclei in the hypothalamus
What is the arcuate nucleus?
Nucleus in the hypothalamus which contain neurones that control energy balance and reproduction
How do tanycytes monitor the bodies internal environment?
- The tanycytes that project to the median eminence are in physical contact with fenestrated capillaries meaning it can sample the blood and look at the internal state
- Also sits next to the ventricle which is full of cerebrospinal fluid which is also full of signals about the internal body conditions
- The hypothalamus can then act accordingly
- The hormones that have been released into the blood can then act negatively back on the tanycytes to stop its release
Why is thought that tanycytes could be stem cells?
Tanycytes look like radial glial cells, look like they could be in a niche with blood capillaries (stem cell niches are heavily vascularised)
How can we test if tanycytes are stem cells?
Test of new cells are born in the adult
- Add a marker for S phase using bromodioxyuradine (BRDU). This acts as an analogue for thymine in DNA replication. This new DNA is then labelled so can see if cell division has occurred and therefor new cells are born
- Inject BRDU into third ventricle of a mouse and see if there are any new neurones that have formed when dissect its brain
- Found that there was de novo neurogenesis in the adult hypothalamus
- This happened in response to acute physiological changes and stressed conditions e.g. high fat diet
Does the fact that because tanycytes are dividing and new neurones are forming prove that tanycytes are stem cells?
No
- Doesn’t prove that tanycytes are the new neurons origin
How can you prove that new neurones forming in the hypothalamus are from tanycytes?
Genetic lineage tracing (cre loxp)
How do you make a conditional knockout?
- Genetically engineer the gene you want knocked out so that it has two flox (flanked by lox) sequences on either side of it.
- Introduce this DNA construct into the embryonic stem cells of the mice. The mouse is now carrying this gene in every cell in its body
- Take a second gene that is only expressed in the tissue interested in and clone the cre recombinase enzyme gene downstream to the genes promotor to make a second transgenic mouse
- Bread the two-transgenic mouse together
- The offspring will then remove the gene in cells that express the tissue specific gene as cre recombinase will be activated and cleave at the lox sites
How can cre lox be sued for tissue specific lineage tracing?
- Produce a transgenic mouse that has a gene expressed in tissue of interest upstream to cre
- Make a second mouse with a reporter gene downstream of a constitutive promotor but upstream to the reporter gene (makes the cell blue) add a stop codon to stop transcription of the reporter gene
- Cross the two mice. The stop sequence is recombined out where the tissue specific gene is expressed meaning the cell will appear blue
- These cells descendants will therefore all be blue
Why is tissue specific lineage tracing more effective then injecting a dye?
Because the colour will not be diluted out after each division
What is meant by temporal conditional techniques?
Able to decide then the gene is knocked out as well as in which tissues
How are temporal conditional techniques carried out?
- Cre recombinase is fused to a mutant oestrogen ligand binding domain (ERT2) that requires the presence of tamoxifen for activity
- This means that Cre is only activated when tamoxifen injected into mice
How did Robins et al, 2013 use tissue specific lineage tracing in tanycytes?
- There are different subsets of tanycytes. Specifically interested in ones that express FGF10. These are alpha tanycytes
- GLAST is a gene expressed in this subset of tanycytes
- Fuse CreERT2 downstream to the promotor of GLAST and cross with a transgenic mouse that has a Flox stop codon upstream to a reporter gene. The reporter gene could be lacz or GFP
- When tamoxifen is administered, reporter is expressed in tanycytes
What did Robins et al, 2013 discover from tissue specific lineage tracing of tanycytes?
That tanycytes are neurogenic
- The alpha tanycytes that were investigated can self-renew and give rise to other tanycytes subsets, to neurones and to astrocytes
- Will usually only produce new neurones in response to change in environment
Why doesn’t tissue specific lineage tracing of tanycytes prove that they are stem cells?
Can’t conclude that tanycytes are stem cells because we can’t say that the new cells were formed from one specific tanycyte as the lineage tracing was carried out on a population of tanycytes
What did Robins et al, 2013 do to prove that one tanycyte can give rise to a population of different cell types?
Complementary in vitro studies
- Hypothalamus was dissected out and dissociated into individual cells
- Plate each cell to one well
- If it is a stem cell it must be able to proliferate. After incubation, the cell differentiated to form a neurosphere.
- The neurospheres were tested for stem cell factors.
- Also added other factors to see if it gives rise to different cell types
- Found that there are single cells in the hypothalamus that can self-renew and give rise to different cell types in vitro
What hypothalamic nucleus was found to differentiate form tanycytes?
Arcuate nucleus
Why is the fact that the arcuate nucleus differentiate from tanycytes give evidence to support them being stem cells?
The arcuate nucleus is responsible for controlling food intake (NPY), growth (GHRH) and reproduction (DA)
All these processes need to be able to adapt to changes in the environment
Why is thought that tanycytes derive from the FGF10+ embryonic multipotent hypothalamic progenitor?
Because they express FGF and they proliferate in response to elevated FGF
What is the idea regarding formation of new neurones involved in body homeostasis?
Emerging idea is that new neurones involved in energy homeostasis can be generated in adulthood from an Fgf-responsive/Fgf10-expressing population. This is important in changing life course
What mechanisms have been investigated in the spinal cord that are also relevant in the hypothalamus?
- What happens between the producing-responding cell: Control of Shh spread
- What happens in the nucleus: how transcription of Shh is governed
Explain the Shh mechanism
- Shh binds to the receptor patched and interacts with co receptors: Hhip, Gas1
- Patched represses the activity of smoothened
- Shh is transcribed and released from the cell. It binds to patched leading to the alleviation of the negative repression of transcription in the nucleus
- Smoothened activates the Gli transcription factors that turn on the expression of several genes
- Turns on the transcription of patched itself and other co receptors and downregulates BOC and GAS1. This leads to huge change in the cells ability to respond. This is called ligand dependant antagonism.
What is ligand dependant antagonism?
When a molecule turns on the expression of receptors that are required to respond to it. This leads to huge change in the cells ability to respond
How does ligand dependant antagonism allow for a steep diffusion gradient over a short distance?
- Usually, there would be more Shh molecules then there are patched receptors meaning that Shh can diffuse to other cells and activate transcription in those cells.
- However, Shh activates transcription of more patched receptors. (ligand dependant antagonism)
- The more molecules of patched on neighbouring cells, the less likely it is that Shh will diffuse to far away cells meaning they won’t be activated.
- This leads to non-cell-autonomous Shh pathway inhibition in cells distal to the Shh source.
How would happen if ligand dependant antagonism stops?
If a set of cells stops expressing Shh and Shh receptors then ligand dependent antagonism is stopped in that region. This will support the continual spread of Shh into neighbouring territories. Because Shh induces its own expression, this will support a spread of Shh expression to the neighbouring territory
How does ligand dependant antagonism play a role in Shh patterning of the hypothalamus at the mRNA level?
- BMP antagonists induce genes such as Pou and Sox which are transcription factors that bind to enhancers and turn on neural genes. These genes (pax7) will then be expressed throughout the ventricular zone. This activates many downstream transcription factors
- Shh expressed from notochord and floor plate which activates homeodomain transcription factor. This GliA- activated homeodomain TF binds to the enhancer of the neural genes and acts as a repressor, stopping the transcription of pax7.
What is SBE2?
An enhancer element for Shh in the hypothalamus
How was SBE2 discovered?
Doug Epstein found that transcription of Shh gne is regulated by a different enhancer element in the hypothalamus then it is in the future spinal cord/hindbrain and midbrain
- Found enhancer element called SBE2. If mutated this element then it lead to regulatory issues of Shh in the hypothalamus
What is the role of Tbx2 in the expression of Shh in the hypothalamus?
Normally, GliA and Sox2 is required for Shh expression
- However, when Tbx2 protein is present in cells, it binds to the SBE2 enhancer causing the displacement of Sox2. This means that transcription of Shh ceases.
Explain the changes expression of Shh that occur during the development of the hypothalamus?
- Shh from early prechordal mesoderm induces GliA and therefore induces Shh transcription
- Later in development, BMP7 is expressed from the prechordal mesoderm and induces Tbx2. This therefore downregulates Shh
How can the discovery of how Shh is regulated in the hypothalamus explain how the key hypothalamic progenitor cells are maintained into the adult life?
The current working model
- Shh induces a Shh+ cell at the ventral midline.
- The prechordal mesoderm properties change and begins to upregulate BMP7 which upregulates Tbx2.
- At this point in time, ligand dependant antagonism is still occurring meaning Shh can’t diffuse very far.
- Tbx2 downregulates Shh and in turn downregulates patched, preventing ligand dependant antagonism.
- This allows Shh to diffuse to the next cell along.
- The neighbouring cell is the FGF10+ progenitor cell. Shh can then be induce into that cell reforming the parent progenitor cell
Give two papers that show the importance of early developmental steps in adult formation?
Liu et al (2017)
Liu, et al (2015)
Give a brief overview of Liu, et al (2017)
- There are a number of wake promoting neurones characterised when this paper was published. However, there wasn’t much information on sleep promoting neurones
- A subset of GABA expressing neurones in the ventral zona incerta of the hypothalamus express another homeodomain transcription factor which is activated by sleep pressure
- They project to the hypocretin neurones and inhibit them
How did Liu, et al (2017) show that these Lhx6-positive GABA releasing neurones were sleep promoting?
They conditionally deleted Lhx9 in the adult mouse. Saw that the mice do not sleep
Give a brief overview of Liu et al (2015)?
- Narcolepsy is a disease that causes the patient to fall asleep often
- Patients with this have been found to have very low levels of a neuropeptide called hypocretin
- Test if Lhx9 is involved in hypocretin induction by gain and loss of function experiments
- This paper gives therapeutic applications for treatments for narcolepsy
What methods did Liu et al (2015) use to isolate hypocretin expressing cells?
Make a transgenic zebrafish in which the hypocretin promotor is linked to red fluorescent protein
Isolate cells using FACS machine which separates fluorescent cells from non fluorescent
How did Liu et al (2015) test if Lhx9 is sufficient for hypocretin specification?
Cloned each gene downstream of a heat shock inducible promotor, Hypocretin positive cells appear when the embryos are heat shocked (induce Lhx9 in all cells).
How did Liu et al (2015) test if Lhx9 is necessary for hypocretin specification?
Knockdown of Lhx9 using Cas9
How did Liu et al (2015) test if Lhx9 directly promotes hypocretin expression?
- Double fluorescence in situ hybridisation against Lhx9 and hypocretin on zebrafish embryos one hour after heat shock. Observed Lhx9-overexpressing cells
- Found in another system that Lhx9 turns on the transcription of another gene. In that paper, they worked out the enhancer that Lhx9 binds to. They therefore looked for similar DNA sequences upstream to hypocretin and mutated it to see if it is unable to activate transcription of hypocretin gene
How do the papers Liu et al (2017) and Liu et al (2015) explain how the hypothalamus works?
The hypothalamus consists of paired neurones that have opposing functions and our behaviour is regulated by the balance of these neurones. These papers are examples of this as hypocretin is the neurone responsible for wakefulness
Why do Liu et al (2017) and Liu et al (2015) show the importance of early development in adult function?
The sleep promoting and inhibiting neurones are induced by Shh
What is Lhx1 responsible for?
Responsible for suprachiasmatic nucleus which is the master regulator of the circadian cycle
What are axons?
Axons are long and carry information away from cell body in action potentials. Their termini contain neurotransmitter loaded vesicles and form synapses with the dendrites of other neurons
What are dendrites?
Dendrites are shorter and usually form a dendritic tree allowing the integration of incoming information from multiple other neurones
What is meant by neurones having polarity?
Polarity in this case refers to neurones having different structures at each end
How do the microtubules of axons and dendrites differ?
- Mature axons have highly polarised microtubules all of which are orientated in the same direction with their plus ends facing the axon terminal
- Dendrites also have microtubules but these are less ordered and have mixed orientations
What are microtubule-associated proteins (MAPs)?
Accessory proteins that stabilise microtubules by cross linking the separate strands and inhibiting polymerization
What microtubule-associated proteins are used by axons and dendrites?
Axons - Tau
Dendrites - MAP2
How can axons and dendrites be distinguished between?
The different MAPs can be used as molecular markers to distinguish between axons and dendrites
Give examples of proteins that show the compartmentalisation of the plasma membrane in neurons?
- L1 is a cell surface adhesion molecule that is restricted to axons, whereas the glutamate receptor component, GluR1, is restricted to the cell body and dendrites
- L1, like other axonal components, is added to the axon at the growth cone
What is meant by neurons having an actin dependant diffusion barrier?
Maintains the membrane compartments in the neurones as actin filaments form a meshwork directly under the plasma membrane giving shape and resilience
Why are membrane compartments requires in neurons?
Help to maintain the axons distinctiveness and neuronal polarity
Give evidence for an actin dependant diffusion barrier in neurons?
(Winckler et al., 1999)
- Beads coated with antibodies to L1 cannot easily be dragged (using optical tweezers) across the boundary into the cell body, whereas anti-GluR1-coated beads can be dragged into dendrites from cell body. This shows that there are somatodendritic and axonal domains.
- Other proteins could not be dragged across the axon boundary but could be pulled around the soma and dendrites with ease, showing there is a barrier to diffusion at the base of the axon
- Cell surface molecules appear to be held sections by the underlying actin cytoskeleton
How can optic tweezers be used to produce quantitive data?
The use of optical tweezers can not only be used to track and move beads but can also turn it into quantitative data. This can be done by using the amount of laser movement needed to push the bead to investigate the force to which the bead is bound to the cell surface
Give evidence for how neuronal polarity occurs
Dotti et al., 1988)
- Used hippocampal neurons from the mice and cultured them. They followed how the neurons grew over time
- Initially, the cell is spherical with no projections. The actin filaments under the plasma membrane maintain a barrier to outgrowth
- This barrier is then disrupted and allows the microtubules to enter allowing formation of filopdoia
- These outgrowths are very dynamic, they then expand and form neurites (any projection from cell body).
- These neurites increase in length until one becomes longer than the others and forms a growth cone (becomes polarised)
- This extension will become the axon and may then synapse with other neurons in the culture.
How is a neurite chosen to become an axon?
The cell explores different neurites until one is chosen to be the axon. This choice is considered stochastic (random but may be underlying mechanisms that are not understood)
What happens to neurites that don’t become the axon?
They mature into dendrites
What kind of microtubules are present in growth cones?
Dynamic microtubules
What makes microtubules dynamic?
They are tyrosinated
How are microtubules stabilised?
When an axon starts to form, acetylated microtubules accumulate. These microtubules are therefore stabilised and crosslinked
Give evidence for the importance of microtubule stabilisation in neuronal polarity
- Can artificially stabilise microtubules by adding the drug Taxol to one of the neurites. This forces the choice of neurite to become the axon
- This also suggests that there must be competition between the neurites to stabilise their microtubules and that there may be a feedback loop involved to stop the stabilisation of the microtubules on neurites not chosen
What is required to ensure that only one neurite becomes an axon?
There must be feedback loops - positive and/or negative
Give evidence for the involvement of feedback loops in neuronal polarisation?
- Chop off the axon that has been chosen
- The neurone will choose another neurite to become the axon
- This shows that there must be some sort of negative feedback loop in the axon chosen to supress the other neurites from becoming axons
How could the feedback loop involved in neuronal polarity be positive?
The positive feedback loop could act by upregulating things, that are required for axon formation, in neurites that have already started to become an axon
What are the theoretical models for negative and positive feedback loops?
Positive feedback
- Reinforcing signal that is released from the leading edge of axon
Negative feedback
- Diffusible inhibitor - diffuses through the rest of the cell and prevents another leading edge
- Limiting components – only enough components to form one leading edge
- Mechanical tension – to prevent formation of other leaning edges
Give evidence for the involvement of positive feedback loops in neuronal polarisation?
Overexpression of HRas or PI3K
- Results in multiple axons
- If you add a PI3 kinase inhibitor then you can block the induction of multiple axons showing that HRas activates PI3 kinase
- Also seen that activation of PI3K leads to the activation of HRas – a positive feedback loop
Give evidence for the involvement of negative feedback loops in neuronal polarisation?
HRas is depleted from non-axonal regions
- To begin with, there are no areas that have particularly high levels of HRas protein – evenly distributed
- When the cell becomes polarised, HRas becomes concentrated in the axons and a decrease of HRas in the cell body and other neurites
- This shows a negative feedback loop - a limiting factor
Give the role of PI3 kinase in microtubule stabilisation
- Downstream of many signaling receptors, PI3K elevates PIP3 which phosphorylates Akt.
- This inhibits the activation of GSK3beta.
- GSK3beta normally will destabilize microtubules
- Can see that phosphorylated Akt can be seen in the tip of the growth cone of nascent axons but not neurites
- Inhibition of GSK3beta results in the induction of multiple axons showing the importance of microtubule stabilisation
Give a protein involved in asymmetric division that affects neuronal polarity
Par-3
Give the role of Par-3 in C.elegans
- Through genetic screens, mutations that affect asymmetric division were found
- There are partition defective genes that fails to develop asymmetric divisions. They are required for egg polarity after sperm entry. They Initiate polarisation through the mutually antagonistic interactions of the Par-3 complex with Par-1/2
- Downstream these affect microtubule organisation which in turn differentially localises components of the cell
