Developmental Neurobiology Flashcards
How do Neuroepithelial cells divide?
Symmetrically
How do radial glial cells divide?
Asymmetrically
Injection of BrdU into a pregnant rat at embryonic day 13, followed by analysis of BrdU+ nuclei in the cerebral cortex at postnatal day 2 (P2) shows:
To which cortical layers cells that have stopped proliferating on E13 have migrated
Regarding the extension of Retinal ganglion cells
Sperry’s retinal ablation experiments demonstrated that:
Retinal ganglion cells can extend axons to specific targets in the tectum after optic nerve section
Regarding growth cones & guidance cues
What does Reprogramming of growth cones at intermediate targets allow?
Allows the same guidance cues to be used in different ways before and after the target
Allows growth cones to ignore a cue in one part of the pathway that they responded to previously
What is a key finding from the classic experiment by Hamburger?
The size of the target correlates with the amount of innervation
The Campenot chamber experiment demonstrates that NGF (nerve growth factor)…
-Promotes cell survival at a distance
-Is required for axon/neurite survival
-Promotes axon growth
What is the induction of the NMJ by motor neurons experimentally demonstrated by?
The failiure of NMJs to form in mice in which Agrin is conditionally knocked out in motor neurons
Regarding synapse selection & induction
TRUE OR FALSE?
Contacts made by the growth cone on post-synaptic can initiate synapses
TRUE
Regarding synapse selection & induction
TRUE OR FALSE?
Pre-established specialisations on post-synaptic cells can mark the future synapse, enhancing synapse formation at other sites
FALSE
They can mark the future synapse, however this inhibits synapse formation at other sites
Regarding synapse selection & induction
TRUE OR FALSE?
Axon branches can initiate synapses by random contacts
TRUE
Regarding synapse selection & induction
TRUE OR FALSE?
Dendritic filopodia cannot initiate synapses with passing axons
FALSE
They CAN initiatie synapses with passing neurons
Regarding synapse selection & induction
TRUE OR FALSE?
Differential expression of different members of the neurexin/neuroligin means pre-synaptic neurons can only select their exact post-synaptic partners
FALSE
Differential expression allows pre-synaptic neurons to select between different post-synaptic partners
What types of connection can be made by a single pre-synaptic neuron with different post-synaptic cells
Inhibitory & Excitatory connections
Regarding synapse selection & induction
TRUE OR FALSE?
A single neuron can only receive one type of input (excitatory or inhibitory)
FALSE
Can receive both
Regarding synapse selection & induction
TRUE OR FALSE?
Differential localisation of neuroligins can allow post-synaptic cells to receive spatially seperate innervation from different pre-synaptic partners
TRUE
In mature muscle, each fibre receives input from…
Only a single motor neuron
Synapses are strengthened by…
Co-ordinated activity between pre & post synaptic cells
TRUE OR FALSE?
Motor neurons initially innervate multiple muscle fibres, and each fibre receives multiple inputs
TRUE
What is an MMP
Matrix Metalloproteinase
Regarding MMP as a mediator of synaptic strengthening.
What happens if MMP activity is inhibited?
Reduces synapse co-activity
Low frequency stimulation of the pre-synaptic cell results in a long lasting decrease of post-synaptic response (LTD) because…
-Numbers of AMPA receptors are reduced in the post-synaptic cell
-ProBDNF accumulates instead of BDNF
Regarding the mature phenotype of a neuron
TRUE OR FALSE?
It cannot be varied by factors released by targets that it contacts
It is determined by the set of transcription factors that it inherits from its progenitors
FALSE - It CAN be varied
TRUE
What effect does Regionalisation into distinct progenitor cells have on a neural stem-like cell?
Reduces the potency of the cells
Define differentiation
Process by which cells become different from each other & acquire specialised properties
What is differentiation governed by?
Changes in gene expression, which dictates the repetoire of protein synthesis
What do Neural stem-like cells express (transcribe)?
Genes that regulate the ‘multipotent’ state
i.e. Genes that regulate slow cell cycle or code for proteins that inhibit differentiation
What do Progenitor cells express (transcribe)?
Genes that regulate a faster cell cycle, or code for proteins that direct particular differentiation paths
What do Differentiated cells express (transcribe)?
Genes that regulate cell cycle exit, or code for proteins that enable terminal differentiation
Regarding information guided differentiation
Describe characteristics of Extrinsic information
-Received from cells surroundings
-An ‘undecided’ early cell receives chemical signals from a neighbour
-These alter the receiving cell by changing & restricting its fate
Regarding information guided differentiation
Describe characteristics of Intrinsic information
-Already in the cell
-Provides a ‘memory’ & ‘instruction’ for further fate specification
-Intrinsic information is a type of transcription factor
What is in situ hybridisation used for?
Localisation & detection of specific DNA & RNA sequences in cells, preserved tissue section or entire tissues
It’s used to look at protein signatures
What is immunohistochemistry?
Lab method that uses antibodies to check for certain antigens in a sample of tissue
The antibodies are usually linked to an enzyme or fluorescent dye
How can genetic material (that is identical in every cell) direct differences in cells?
Gene expression can be changed through extrinsic signals or intrinsic information
What is Neural Induction?
In early developing embryo, some ectodermal cells are induced to change their fate & become neural stem-like cells
Cells change to become neural under the influence of secreted factors from neighbouring cells
Briefly describe Generic signal transduction
-Transcription factors cytoplasm -> Nucleus
-Bind to enhancers on promoters on an array of cells
-Change transcriptional signature on that cell
The key pathway for neural induction is the BMP (bone morphogenetic protein) pathway
Describe how this pathway leads to downstream phosphorylation
-External cell makes & secretes BMP
-Diffuses in extracellular space
-Responding cell has BMP receptors
-When BMP binds, activates secondary signal transduction cascade
-Leads to downstream phosphorylation
What is the function of Chordin & Noggin
Proteins that antagonise BMPs - by binding with a high affinity in the ECM to prevent them from binding to BMP receptors
Regarding the BMP signalling pathway
Downstream phosphorylation can end up phosphorylating SMAD 158, what does this cause?
End up with Phosphorylated SMAD 158
-Goes into the nucleus
-Binds enhancers & promoters in different subset of genes
-New transcriptional signature in the responding cell
In the mesoderm, a small group of cells become different to the rest
What do they become?
Where do these lie?
Become dorsal mesoderm / Organiser cells
They lie only next to one region of the ectoderm
What do Ectoderm cells differentiate into?
If close to dorsal mesoderm - Specialised neural stem-like cell
Further way - Skin
Dorsal mesoderm (organiser cell) makes around 10 BMP antagonists
Name 3
What do they all have in common?
Chordin, Noggin, Follistatin
-All mRNAs
-Only transcribed in dorsal mesoderm cells
-Proteins are translated & then diffuse into neighbouring territory
What happens to Phosphorylated SMAD 158 if you lose continued BMP signalling
Lose the phosphorylated SMAD 158 status
What is SOX2?
-Transcription factor that marks neural stem-like cells
-Gene
-Tells cells they will become neural stem-like cells
Where is the neural plate induced?
In the ectoderm where BMP is inhibited
Describe the main aim & type of experiment used by Spemann & Mangold in their ‘organizer graft’ experiment
What did they hypothesise?
-Gain of function experiment to enquire what do organiser cells achieve
-Hypothesised that organiser cells are a source of signals that induce neural fate
What did Spemann & Mangolds Organiser graft experiment results show?
Why?
-End up with twinned embryos
-As you’ve taken the organiser from a donor embryo and grafted it onto the ectopic location of the other
-BMP antagonists end up inducing a second neural plate
Regarding Spemann & Mangold’s organiser graft experiment
How did researchers identify that BMP antagonists were responsible for inducing neural tissue
-All mRNA from organiser was extracted
-It was then reverse transcribed to cDNA
-These were introducted ectopically to look for a gene that would mimic the organiser’s ability to induce a secondary neural plate
Define neurulation
A process in which the neural plate bends up and later fuses to form the hollow, neural tube that will eventually differentiate into the brain and spinal cord of the CNS
Describe the features of ‘neural inducers’ in organiser cells
-Must be expressed in organiser
-Must be secreted & act on adjacent cells
-Overexpression of molecule in an ectopic site should lead to the induction of a secondary axis
-Inhibition of activity should prevent axis formation
What drives neurulation?
Describe this process
HINT - G
-Transforms embryo…
-The germ layers
Gastrulation
-Transforms embryo from 1-D layer of cells into a multi-layered embryo in which the adult body plan is recognisable
-The germ layers assume their final positions & the axes become obvious
Organiser cells have specialised transcriptional signatures (e.g. BMP antagonists aswell as Siamois & Gsc)
Describe what the transcription factors Siamois & Gsc do
-Transcriptionally activate many genes
-These ^ Encode factors to direct the organiser cells’ next step
Organiser cell differentiate into axial mesoderm
What is axial mesoderm composed of?
Which ones move inside first?
Prechordal mesoderm & Notochord
Prechordal cells move in first, Notochord after
Where does the Prechordal mesoderm lie?
Beneath the future forebrain
Describe the structure of the axial mesoderm
Include:
Nervous system
Rod of axial mesoderm
Neural plate
Mesoderm
Endoderm
-Nervous system on top
-Rod of axial mesoderm underlies the midline of the induced neural plate
-Mesoderm drags layer of endoderm underneath it
Describe the action of neurulation
What is it driven by?
What can cause this?
HINT:
-Purse string
-F actin
Driven by changes in cell shape
-A band of F actin is at the apical domain
-IF it pulls tight, the cells are constricted at that edge and so change shape (purse string)
-Many molecules are differentially distributed to either basal or apical edges
-Many molecules interact with F actin to make it constrict
Regarding major types of cell movement that occur during gastrulation
Describe Invagination
Sheet of cells bends inwards
Regarding major types of cell movement that occur during gastrulation
Describe Ingression
Individual cells leave an epithelial sheet and become freely migrating mesenchyme cells
What are mesenchyme cells?
Loosely organised embryonic connective tissue of undifferentiated cells that give rise to most tissues
Regarding major types of cell movement that occur during gastrulation
Describe Involution
An epithelial sheet rolls inwards to form and underlying layer
Regarding major types of cell movement that occur during gastrulation
Describe Epiboly
A sheet of cells spreads by thinning
Regarding major types of cell movement that occur during gastrulation
Describe Intercalation
Rows of cells move between one another, creating an array of cells that is longer but thinner
Regarding major types of cell movement that occur during gastrulation
Describe Convergent extension
Rows of cells intercalate, but the intercalation is highly directional
Regarding Anterior-Posterior regionalisation
Which part becomes which of the CNS
Anterior becomes brain
Posterior becomes spinal cord
What are the key transcription factors for:
-Brain progenitor cells
-Spinal cord progenitor cells
Brain - OTX2
Spinal cord - HOX
What do prechordal mesoderm cells transcribe & secrete? (Notochord cells don’t)
HINT: Signalling pathway
Wnt antagonists (Dikkopf/Dkk)
(Wnt is a signalling pathway)
Describe how Wnt signalling effects the development of the nervous system
High Wnt signalling - Becomes spinal cord
Mid Wnt signalling - Becomes midbrain
Low Wnt signalling - Becomes forebrain
Develop a gradient where posterior end has lots of Wnt signalling and this decreases anteriorly becuase of the Wnt antagonists
How can we tell that the neural plate is becoming regionalised?
Look at the expression of transcription factors
-Using in situ hybridisation - to detect mRNAs
OR
-Using immunohistochemistry - to detect proteins
Describe how loss of function studies show the importance of Hox genes in conferring regional identity:
Part of hindbrain are characterised by Hoxa1 & Hoxb1
If these 2 genes are knocked out, these regions of the hindbrain never form
What is the use of Retinoic acid during development?
Induction
Motor
N P
Involved in:
Induction of neural differentiation
Motor neuron axon outgrowth
Neural patterning
What are neural plate border cells?
What do they do?
-Cell that borders neural plate
-Seperate neural plate from proper epidermis
-Generate neural crest cells
What does the Neural crest cell do?
Generates the PNS
Why are a specialised set of border cells at the edge of the neural plate important?
R p f & d n t p
-Neural crest formation & PNS development
-Roof plate formation & dorsal neural tube patterning
-Closure of the neural tube
Neural crests are known as the 4th germ layer as they give rise to a huge amount of different cells in the body including…
Which of these are from the Ectoderm, which from Mesoderm?
E: M, S c, N
M: O, A, C
From ectoderm:
-Melanocytes
-Schwann cells
-Neurons
From Mesoderm:
-Osteoblasts
-Adipocytes
-Chondrocytes
What is the function of Roof plate cells?
Organising centre for surrounding neuroepithelial cells, promoting their proliferation & specfication
What is a morphogen?
-Molecule that provides positional info by establishing a gradient that is translated into discrete fates
-Secreted, diffusible molecules
-Generate several cellular states in response to different thresholds of concentration gradients
-They are instructive and act in a direct manner
At the time of the neural plate/early neural tube formation, where can the axial mesoderm be located?
Lies just below the ventral midline of the neural tube
As the axial mesoderm forms it begins to express a new gene
State the name and briefly describe this gene
Sonic hedgehog (Shh)
-Secreted ligand & morphogen
-Patterns early neuroepithelial progenitor cells
TRUE OR FALSE?
Specific subtypes of differentiated neurons arise in a distinct, predictable zone along the D-V axis of the neural tube
TRUE
Describe how BMP & Shh pathways interact
-They antagonise each other
-BMP antagonists are still expressed in the organiser-derived axial mesoderm
-Together, the opposing gradients of BMPs & Shh pattern the D-V axis
What causes neural cells to differentiate into floor plate cells?
What do floor plate cells do?
-High concentrations of Shh cause Neural cells to differentiate into floor plate cells
-Floor plate cells occupy the ventral midline of the neural tube & themselves come to turn on Shh
TRUE OR FALSE
All progenitor cells become neurons
FALSE
Some stay as progenitors next to the lumen, in a region called the Ventricular zone
Progenitor cells give rise to 3 cell types of the nervous system
Name them
Neurons
Glia
Astrocytes
Describe the location of the Nucleus relative to the Lumen:
During G1 & S phase of the cell cycle
At M phase & Cytokinesis
G1 & S - Away from the lumen
M & C - Close to the lumen
Describe the products of asymmetrical division of Radial glia cells
One daughter - Like it’s mother
Other daughter differentiates into a neuron, migrates away from Ventricular zone
Where do Proliferating progenitors move to?
Which transcription factors assist this?
Move into the adjacent mantle zone
Nkx & Pax are transcription factors which pick up these cells
TRUE OR FALSE?
Notch signalling is an example of lateral inhibition
What is lateral inhibition?
TRUE
Lateral inhibition - The capacity of excited neurons to reduce the activity of their neighbours
What is neurogenesis?
Why does it occur?
Process by which new neurons are formed in the brain
Occurs becuase asymmetric cell division leads to daughter cells with different levels of Notch signalling
What is the overall function of Notch?
Controls the number of cells that form neurons
What are the 2 classes of migration of neurons?
Tangential & Radial
Briefly describe the first part of Radial migration
Interkinetic Nuclear migrations in the neuroepithelium
Briefly describe the second part of Radial migration
Establishment of layered structures, such as the cortex & cerebellum
Regarding Radial migration
Describe why early neuroepithelium is ‘pseudostratified’
Due to migration of mitotically dividing nuclei
-Has appearance of multiple layers because the nuclei of individual cells are migrating up and down
The neurepithelial cells go through different phases of the cell cycle.
Describe what happens to the nuclei during:
G1
G2
Describe where these happen:
S phase
M phase (division)
G1 - Nuclei move up
G2 - Nuclei move down
S phase - Occurs at pial (basal) surface
M phase (division) occurs at the ventricular (apical) surface
What determines the localisation of asymmetric localised cytoplasmic components in radial glial daughter cells?
The plane of cell division
Who’s gonna nail this exam?
Kyan
Regarding Radial migration (part 2)
Describe Altman & Bayer’s experiment using birth-dating to follow neurogenesis & migration
-Triated thymidine incorporated into newly synthesised DNA
-Inject this DNA into pregnant female rats, it incorporates into cells in the S phase
However only those cells in their final division retain the label over time
-This ‘birth-dates’ the cells, allowing us to trace their migration to their final destination over time
Regarding Radial migration, describe the key finding from Altman & Bayer’s birth-dating experiment
What can we draw from this?
Noticed that neurons ‘born’ at different times migrated to different layers of the cortex
Each layer of the cortex is characterised by the expression off a specific set of transcription factors, indicating that neurons born at different times have different fates
Regarding Cortical neuronal fates changing over time
Describe the Classical ‘heterochronic’ transplant test
HINT - Whether the fates of neuronal precursors at different ages is fixed or plastic
Early precursors
-Transplanted to older host
-Migrate to & Adopt fate of cells being born at this time in the host
-Their fate is still plastic
Late precursoers
-Transplanted to younger host
-Migrate to & adopt fate of the position they would have had if they hadn’t been transplanted
-Potency is lost
Suggests early precursors have the ability to adopt many fates, but that is lost in age
Suggests that for early precursors, their might be something in the environment that influences their fate
Describe Lissencephaly
-Smooth brain
-Majority of neurons found in deeper layers
-Should be distributed throughout the lobes
-Due to failure/abnormality of neuronal migration
What can cause Lissencephaly (smooth brain)
-Mutations in genes of proteins associated with microtubule function, such as:
-TUBA1A
-TUBB2B
-LIS1
-DCX
What is the function of Cajal-Retzius cells?
Tell migrating cells when to stop
Describe how the ability to visualise Cajal-Retzius cells with GFP is beneficial to us?
Allows us to see that they change shape & die in postnatal period
Describe the experiment used to tell us what Cajal-Retzius (C-R) cells do
HINT - Analysis of the Reeler mouse mutant
-Mutation is in Reelin gene, encoding large ECM protein expressed by C-R cells
-Loss of Reelin leads to the failure of C-R & subplate cells seperating
-Consequent disruption to layering of cortex
-Thought to be because of the absence of Reeler protein, it causes migrating neurons to fail to stop
In humans, what do Reelin mutations lead to?
Lissencephaly (smooth brain)
Describe what happens to the following cells over time:
Radial glia
Progenitor
Mature neurons
Intermediate progenitors
Radial glia - Deplete over time
Progenitor - Decreases, very few left by birth
Mature neurons - Increases
Intermediate progenitors - continue to proliferate in sub-ventricular zone & produce upper layer neurons, even these decrease & disappear completely
Briefly describe the importance of Radial glia
-Expand neuroepithelium
-Expand size of the cortex
-Make many of the cells of the cortex
TRUE OR FALSE?
Adult neural stem cells are a subset of Astrocyte cells
FALSE
They are a subset of Radial Glia cells
What are the 2 main types of neurons of adult neural stem cells?
Where do they go?
What do they do?
Olfactory neurons
-> Subventricular zone of 4th ventricle
-Migrate via Rostral migratory stream to the olfactory bulb
Granule neurons
-> Granular layer (Dentate gyrus) of hippocampus
-Involved in spatial memory
Regarding Tangential migration
How is the forebrain patterned?
In the dorsal-ventral axis
By BMPs, Shh & other factors
What is the subpallium?
The source of many interneurons for the cortex & other regions of the developing brain
Where do inhibitory interneurons of the cortex migrate FROM?
The Subpallium
Describe where the following interneurons go:
GABA-ergic Inhibitory interneurons
Dopaminergic Inhibitory interneurons
Cholinergic inhibitory interneuons
GABA -> Cortex
Dop -> Olfactory bulb
Cho -> Striatum
Name the 3 layers of the cortex of the cerebellum
From Highest to lowest
Molecular layer
Purkinje cell layer
Granule cell layer
Where are major cerebellar neuronal types derived from?
Rhombic lip of the developing hindbrain
Describe Rhombic lip cells
-Like neural crest cells
-Highly proliferative
-Have migrating, proliferative daughters
Describe how the following migrate & what they do
Anterior Rhombic lip daughter cells
Posterior Rhombic lip cells
Granule neuron precursors
Anterior Rhombic lip-
-Migrate tangentially across surface of anterior hindbrain
-Form External Germinal layer of cerebellum
-Later differentiate into granule neurons
Posterior Rhombic lip-
-Migrate tangentially to ventral hindbrain
Granule neuron precursors-
-Proliferative zone on the outside (pial)
-Tangential then Radial migration
Regarding Cerebellar disorders & disease
Lissencephaly in the cerebral cortex is caused by Mutations in Reelin, this can also display cerebellar hypoplasia
Discuss how Mutations in Reelin can lead to cortical & cerebellar disruptions
Reelin is expressed by granule neruons above & below the Purkinje cell layer
Loss of Reelin leads to disruption of Purkinje cell layer
Mutation in Reelin receptor (VLDLR) can also cause disruptions
Describe Sperry’s Chemoaffinity Hypothesis which explains how specific neuronal connectivity of the adult organism arises
Directed & Specific outgrowth occurs through axons following ‘individual identification tags’ carried by the ‘cells & fibres’ of the embryo
What are Guidance cues?
Factors that axons can use to find their correct targets
Relatively early experiments to identify the location of Guidance cues was done in insects
Why?
Simple Nervous system
Embryos easy to observe & manipulate
Individual cells could be ablated
Explain why G axon ‘stalls’ in the absence of P axon
-NOT due to lack of axons on which to extend
-NOT due to reduction in numbers of axons
-DUE to G axon growth cone looking for specific cues on the P axon
What does the Labelled pathway hypothesis tell us?
-Axons can selectively fasciculate with others
-Axon surfaces carry labels/cues
-Different axon growth cones express different sets of receptors for such cues
-Early axons (Pioneers) form an axon scaffold on which later axons (Followers) can extend
How did Pioneers (Early axons) know where to go?
Followed stereotyped paths
Growth cones appear to react at specific points in the pathway
There must be molecular differences in the environment
Describe some experimental evidence showing the work of axon guidance cues
Cell ablations lead to growth cone stalls if an attractive force has been lost
Ti1 growth cone seems to avoid the limb boundary, as if it were inhibitory
What are the 4 forces of axon guidance?
Contact attraction
Chemoattraction
Contact repulsion
Chemorepulsion
Discuss the similarities and differences between Lamella & Filopodium
-Made up of different kinds of F-actin
Lamella - Actin bundles crosslinked into a net
FIlopodia - Actin bundles are polarised to form larger bundles
Neither are stuck down, they are highly motile
In the resting growth cone, tubulin is dragged sporadically into the filopodia
What do we need to make this happen more dramatically?
Growth cone coming into contact with an attractive cue
TRUE OR FALSE
Growth cones don’t turn, they reorganise
TRUE
During development, if a cell has commited to a particular fate, it is said to be:
Determined
The initial dorsal-ventral axis in amphibian embryos is determined by:
The point of sperm entry
What is the correct receptor to be paired with the ligand FGF?
Receptor tyrosine kinase
What is the correct receptor to be paired with the ligand Shh?
Patched
What is the correct receptor to be paired with the ligand Wnt?
Frizzeld
The Phenomena that some cells evoke a specfic developmental response in other cells is known as…
Embryonic induction
Chordin is homologous to the Drosophila protein:
sog
TRUE OR FALSE
Shh is capable of instructing a cell to take at least 2 seperate fates
TRUE
As Shh is a secreted morphogen
Neural inducing molecules from the early organiser…
Induce neural tissue that is anterior in character
HIndbrain serotonergic neurons (FLIP ME)
Originate from progenitors that may be patterned by the retinoic-Hox code
Originiate from progenitors that are induced by Shh
Differentiate as they migrate along a radial glial process
Differentiate as they upregulate Notch signalling
