Neural Stem Cells, Neural Tube Development and Migration

Question 1

What are neural stem cells?

Answer 1

Neural Stem Cells are stem cells of the nervous system that generate neurones and oligodendrocytes and astrocytes in the embryo and the adult CNS. Microglia come from common myeloid progenitors (not NSCs).

Question 2

What forms the neuroepithelium?

Answer 2

In the embryo, a neural plate develops, which then subdivides to brain, spine; then further to forebrain, midbrain, hindbrain and different spinal levels. The ectoderm cells that form the neural tube are called neuroepithelial cells.

Question 3

What general processes do neuroepithelial cells go through to form the adult brain?

Answer 3

The neuroepithelial cells go through a process of proliferation. Interesting to a evolutionary perspective, small tweaks in the rate of proliferation lead to greater number of neurones –> greater cognitive ability –> the basis of human capacity for social interaction, language and society.

After proliferation, the neuroepithelial cells start to specialise by responding to signals from four axis. The pattern of these signals with the timing of each, tells each cell where it is, and thus what cell to produce. Before a certain time, you can move around these stem cells to different locations and see them differentiate into appropriate cells. After a certain time, their genomes become restricted, and so can only generate certain populations even after transplanting in different locations.

Radial glial cells were thought to only be scaffolds, however they are stem cells themselves all over the brain. Elegantly their daughter cells use the radial glial cells to travel to where they are needed.

Lots of neurones are overproduced. It doesn’t matter as they compete for survival to form circuits.

Question 4

What are the main properties of stem cells?

Answer 4

Properties of stem cells:

1. Divide
2. Self-renew
3. One or more of the daughter cells can become many different cell types

Question 5

What are the potency types stem cells?

Answer 5

Totipotent stem cells exist for 4-5 days after fertilisation. The genome is completely open, but the cells are hard to work with in a lab. Note: totipotent stem cells can also become placental cells.

Embryonic stem cells (ES) Can’t make placenta or outside of embryonic tissue but can differentiate into all 3 germ layers.

Induced pluripotent stem cells (iPS/iPSCs) are artificially made and have potential for many therapeutic uses.

Embryonic or adult tissue specific stem cells are multi or unipotent.

Totipotent – all cell lines, including extra-embryonic material
Pluripotent – all embryonic cell lines
Multipotent – reduced potency
Unipotent – can only make one cell type

Question 6

Describe the features of embryonic stem cells?

Answer 6

The preimplantation embryo (‘blastocyst’) contains totipotent cells. At day 4-5 after fertilisation, there exists cells that allow implantation into the womb as well as cells that will become the inner cell mass (‘ICM’). Embryonic stem cells, are cells are in the ICM. They can self-renew indefinitely (in culture) and are pluripotent (can differentiate into all 3 germ layers). However, the embryo only contains pluripotent cells for a short while as cells start to become committed to lineages. Hard to work with in a lab.

Question 7

What are three germ layers, and what do they form?

Answer 7

The 3 germs layers:
• Endoderm - internal layer - forms e.g. lung, thyroid, pancreas
• Mesoderm - middle layer - forms e.g. cardiac muscle, skeletal muscle, kidney tubule cells, red blood cells, smooth muscle
• Ectoderm - external layer - forms e.g. skin and neurones

Question 8

What are the pros and cons using embryonic stem cells in the lab?

Answer 8

ES cells are useful because they can:
• Can be stably maintained and expanded in vitro
• And driven to differentiate into particular cell types

Research into human ES cells presents ethical issues as it almost always involves destroying a preimplantation embryo. ES are licensed in UK for embryos created in vitro (up to 14 days). Law varies across Europe. USA restricts human ES cell research (to only existing lines or state or privately funded). This pushed for a strong drive to find alternative human pluripotent cells.

Question 9

What cocktail of transcription factors can induce multi-potent

Answer 9

Introduced 4 transcription factors: Oct3/4, Sox2, c-Myc, and Klf4 into mouse embryonic or adult fibroblasts (i.e. differentiated somatic cells).The resulting cells behaved like ES cells (in many ways) and were called Induced pluripotent stem cells (iPS/iPSCs).

Question 10

What are the two main advantages of using iPSCs

Answer 10

Skips ethical issues + can be used from patient’s own cells!

Question 11

What are the two zones in the post-natal adult brain that can make new neurones?

Answer 11

Postnatal and adult mammalian brains can make new neurones
• This was a dogma that had to be challenged, though we know more about rodents than humans
• Most of the brain will resist cell division, this is to prevent cancer. There are 2 main regions where there is exception to this rule:
○ Striatal subventricular zone (SVZ)
○ Subgranular zone (SGZ) of the hippocampus

Question 12

Describe the stem cells of the Sub-ventricular Zone (SVZ)?

Answer 12

These stem cells are a type of astrocyte (contain biomarkers GFAP+ and Prominin-1+ which are normally found in astrocytes). They are in the ependymal or subependymal layers (both contacting the CSF). Ependymal cells waft the CSF.
• They are kept quiescent/paused at dividing (G0 of cell cycle).
• Can divide asymmetrically (into stem cell and differentiated one).
• Produce ‘transit-amplifying cells/progenitors’, Type-C cells - daughter cells that can divide lots of times, instead of mother cells. This has a lower cancer risk.

Question 13

What cells do the stem cells in the SVZ produce?

Answer 13

In rodents, the type-C cells divide to give neuroblasts (type-A cells) PSA-NCAM+ (identified with that neuroadhesion molecule).They then migrate to olfactory bulb via rostral migratory stream (RMS) in rodents. They also generate oligodendrocytes.

In humans, the SVZ neuroblasts produce striatal interneurons (which seem to be depleted in Huntington’s disease).

Question 14

Describe the stem cells of the subgranular zone.

Answer 14

These are a type of astrocyte/radial glia-like cell (GFAP+, nestin+ and Sox2+).
• Quiescent, can self-renew & generate radial-glia like cells, neurons and astrocytes (some say subtypes have varying potency).
• The neurons formed are glutamatergic dentate granule cells

Question 15

Describe the function of the stem cells in the subgranular zone.

Answer 15

There is neurogenesis in the human adult hippocampus SGZ. [Paper: “Dynamics of Hippocampal Neurogenesis in Adult Humans” - Spalding et al., 2013]. About 700 new hippocampal neurons per day are produced.

Why bother to make new neurones in the hippocampus if it opens us up to cancer risk? Functions of the neurones generated from the SGZ include:
• Spatial learning, fear conditioning, clearing ‘memory traces’, navigation and depression
• (Antidepressant mechanism?)

Question 16

How do Adult NSCs know when to proliferate?

Answer 16

Neuronal activity nearby can stop SGZ NSC proliferation. This occurs via GABA ‘spill-over’ release from interneurons (Song et al., Nature, 2012). Some of the GABA neurotransmitters leak out of synapses. Hippocampal NSC detect this background GABA leakage - if it stays high, they stay quiescent. If it falls low (i.e. there has been cell death and hence neurotransmitter leakage), they respond and proliferate.

Question 17

What are the steps in making induced pluripotent stem cells?

Answer 17

1. Take skin biopsy, put in culture and separate out the cells.
2. Develop fibroblast monolayer
3. Add 4 transcription factors: Oct4, Sox2, Myc and Klf4
4. Wait for reprogramming (2-3 weeks)
5. Colonies of piled-up cells will develop into iPSC colonies

Question 18

How do you check that the iPSC created are actually pluripotent?

Answer 18

Promoters and histones of pluripotency genes will be demethylated (i.e. active)
Chimeras: The iPSCs (e.g. labelled with GFP [green fluorescent protein]) can contribute to an embryo (microinject the cells into blastocyst, + pseudo- pregnant mouse, and trace later, can contribute to germ line)

Question 19

What is Nanog?

Answer 19

A test for pluripotency.

Question 20

How can you use iPSC for in the research of ALS?

Answer 20

• Taking a skin cell from a patient, inducing it into a PSC then doing genetic sequencing to look for mutation responsible.
• Model the disease. It is possible to differentiate these iPSCs into NSCs then neurones. Take embryoid bodies formed from the iPSCs then treat with a Shh and RA agonist, grow on laminin. The cells differentiated into motor neurons and expressed MN-specific transcription factors.
• These motor neurones can be used to test different drugs on via iPSCs. Regarding ALS, none worked, but was important for research, it showed results quickly and didn’t involve human participants, which could have been catastrophic.

Question 21

Outline CNS Development

Answer 21

• Part of the embryo is ‘designated’ to become the future CNS, rather than skin.
• Cells divide (uncommitted, pluripotent for a few days at least). There is a period of rapid division.
• Some regionalisation: signalling centres develop. This involves coordinating with surrounding tissue.
○ There are four main axis: top-tail, back-front, left-right, time.
• Cell mixing restricted (usually by cell adhesion molecules) once a stem cell becomes committed.
• Neurones start to be generated
• Migrate to final positions
• Then produce glia and more neurones. Neurone producing genes get turned off, then astrocyte or oligodendrocyte genes are turned on - often by the same precursor stem cell.
• Connect and generate synapses
• Cull excess as lots of neurones are overproduced.
• Myelination
• Remodel as required

Question 22

Describe the patterning produced by the mesoderm in differentiating the ectoderm

Answer 22

FGF (fibroblast growth factor) and BMP cooperate to pattern the neural plate (morphogens). BMP4 (bone morphogenic protein 4) in ectoderm is pro-epidermis and anti-neuralising –> important in skin development. BMP and WNT signalling needs to be blocked for neural ectoderm development.
○ Epidermal = high BMP4, low FGF
○ Borders = medium BMP4 and medium FGF
○ Neural ectoderm = 0-low BMP4 and high FGF (FGF leads to SMAD phosphorylation and less to nucleus)

Question 23

Describe the process of neurulation

Answer 23

As the notorchord (part of the mesoderm) develops, it induces the overlying ectoderm at the midline to thicken and to form an elongated plate of thickened epithelial cells called the neural plate. Notochord tells the embryo where the middle is and which is left and which is right. Notorchord leaves quite early in humans (actually now though to tranform into the nucleus pulposus of vertebrate disks). It is initially a good source of Shh, which determines the population.

The neural plate invaginates to form a neural groove and eventually the neural folds meet. This results in the formation of the neural tube, cranial placodes and the neural crest (which will become the PNS), and epidermis (which will become skin).

Question 24

How does rostral-caudal patterning occur in the neural tube?

Answer 24

Rostral-caudal CNS patterning occurs mainly through Wnt signalling. Wnt antagonists clear Wnt from head end, while Wnt is expressed at caudal end. Later Wnt has different roles; Wnt signalling is a huge field, because Wnt has a large variety of roles often seemingly counteractive, but due to different temporal and receptor expression.

Question 25

What condition occurs when the neural tube does not close?

Answer 25

Neural tube defects such as spina bifida are some of commonest and most severe disorders of the foetus & newborn (1-2/1000 live births. Different regions of the neural tube fail to close (neural crest doesn’t join). We used to think the neural tube just folded from head to tail, but there seems to be different regions of closures.

Question 26

How are the neural stem cells specified to become certain progenitors?

Answer 26

In general, the transcription factor profile when the cell leaves the cell cycle will determine the cells fate. The cells leave the cell cycle completely (not into G0, but out of the cell cycle completely) - become post-mitotic - this is also called the cell’s “birthdate”. It will never re-enter the cell cycle unless in very rare exceptions.

Question 27

What causes further development of neural tube cells into neural cells?

Answer 27

Proneural genes are involved in the further development of neuronal progenitor cells - they tell the cell not only are they neural, but sometimes what type of neurone they will become. They code for bHLH (basic helix-loop-helix) transcription factors which are a family of 20+ transcription factors in vertebrates.

bHLH TFs can have different roles at different stages. Several oscillating bHLHs can control multipotency. When stable, they can drive differentiation. We used to think that when a gene is on, it leads to a particular effect, but in reality is it very time (and therefore environment) depended.

Question 28

What proneural gene is found in the brain and spinal chord?

Answer 28

Neurogenin

Question 29

Describe the key signalling centre between the midbrain and hindbrain

Answer 29

Between the midbrain and hindbrain there is a key signalling centre that we know quite a lot about. It is called the isthmic organiser (IsO). The IsO tells cells rostral to it that it is going to be midbrain and not hindbrain, and visa versa. This all occurs via turning on of genes and transcription factors.

Question 30

What key signals drive midbrain development?

Answer 30

In midbrain development key signals include: Wnts, FGF8, Shh. Wnt1 (ventral midbrain) and FGF tell the neural tube which region of the brain it is, and therefore promote specific cell types to develop.
Regions are separated and determined by the factors expressed, can be manipulated by scientists to change developmental regions. Development of the dopaminergic neurones in the midbrain is regulated by a distribution of Sonic Hedgehog (SHH) mutagenic factor which tells the area they are in the ventral part. While the isthmus tells the cells they are in the midbrain.

Question 31

Describe the development of dopaminergic neurones

Answer 31

Development of the dopaminergic neurones in the midbrain is regulated by a distribution of Sonic Hedgehog (SHH) mutagenic factor which tells the area they are in the ventral part (as it is secreted by the floor plate - I think). While the isthmus tells the cells they are in the midbrain (via Wnt1 and FGF). The combination of these produces DA neurones. Two key dopaminergic pathways formed:

1. Nigrostriatal – SNpc to dorsal Striatum - important in making movement; degenerated in PD
2. Mesocorticolimbic – VTA to ventral striatum, limbic system, hippocampus, prefrontal cortex. Important in cognitive emotion including reward, drug abuse, working memory 

Sonic hedgehog in (Blue) Wnt1 is Purple (Isthmus).

After proliferation of the DA progenitors, radial glia aid in migration of the neurones from the ventricular surface of the neural tube to the ventral surface.

Question 32

Describe the role of the cytoskeleton in migration

Answer 32

The cytoskeleton is very important in transporting materials to where the neurone needs it. Microtubules form strong lines where materials such as mitochondria can be transported down to forming synapses.

Actin, is not very strong, but can be assembled very quickly, and so is useful in making filopodia and lamellipodium in the growth cone. In neuronal migration, cells will move my assembling and disassembling the cytoskeleton; the growth cone will grow towards growth signals.

Question 33

Describe the layers of the neocortex.

Answer 33

The neocortex has 6 horizontal layers of neurones. Most of the neurones (80%) are excitatory projection (pyramidal) neurons (usually glutamatergic). These tend to have long axons and distant synapses. Some layers have been well mapped:
• E.g. Layer 5 connects to the basal ganglia, diencephalon, midbrain, hindbrain, and spinal cord

Layer 1 = molecular layer
Layer 2 = external granular layer
Layer 3 = external pyramidal layer
Layer 4 = internal granular layer
Layer 5 = internal pyramidal layer
Layer 6 = Multiform layer

Question 34

Describe cell migration during cerebral cortex development

Answer 34

The telencephalic wall (cortex precursor) is 1 cell thick initially. Large amounts of replication are required to populate this region with neurones. This division is symmetrical (in terms of mitosis) when reproducing the stem cells. Later on, when neurones are required, the daughter cells must become asymmetrical.

Cortical projection neurones are generated from the neocortical neuroepithelium - ventricular zone [VZ] and subventricular zone [SVZ] - ventricular zone is the part of the neuroepithelium that is adjacent to the ventricles (inner part of the tube).
• Retinoic acid (RA) has a key role - i.e. need to pattern the neuroepithelium
• Asymmetric cell divisions from cortical primary progenitors (radial glia) in the VZ generate either an immature projection neurone, or a secondary progenitor (intermediate progenitor in the SVZ)
• Secondary progenitors also generate neurones.
• An immature neuron is generated; it migrates radially towards the pial surface along radial glial processes (Rakic) then settles in specific cortical layers by sensing local molecular cues e.g. reelin

The cortex develops ‘inside-out’: You develop one layer, then the next layers need to crawl on the previous layer to reach the outermost layer. I.e. layer VI developed first, then V, and so on.

Question 35

Describe the role of Cajal-Retzius cells in the context of cerebral cortex development

Answer 35

These are found at the pial surface. They have reelin, a molecule implicated in a range of signalling pathways important in neuronal migration. It is thought that the Cajal-Retzius cells allow meninges to drive migration. Reelin is important for migrating neurones to know where they are going - guidance factor.

Question 36

Describe the role of Radial Glia Progenitors in cerebral cortex development?

Answer 36

Radial Glia also produce neuronal progenitors - they aren’t just for scaffolding for neuronal migration, they can also produce neuronal progenitors. Note not all migration is radially: Some progenitors migrate tangentially (non-radial migration). Some migrate from far away areas - e.g. GABA stellate cells come from the lateral ganglionic eminence [see right].

Question 37

Where are cortical interneurones produced?

Answer 37

Most cortical interneurones are generated outside of the cortex (humans generate a few in the cortex). Most actually develop in the primordium of the basal ganglia - called the ganglionic eminences. Then tangentially migrate into the immature cortical plate. Some GABAergic interneurons remain in the basal ganglia (e.g. striatal interneurons).

Question 38

Describe the features and fate of Neural Crest cells

Answer 38

NC houses the vertebrate multipotent stem cell population. (Not just neural cells, but also skeletal and other tissue types). The cells leave the dorsal CNS via EMT (epithelial to mesenchymal transition) - so can become more mobile and spread out. Lose epithelial adhesions; lose cell polarity; and cytoskeletal remodelled. Snail/slug and twist are important genes for this. The neural crest cells:
• Migrate widely
• Have diverse fates. E.g. some become the jaw, smooth muscles, melanocytes, cranial nerves, thymus etc.
• The cells become restricted as they migrate.

Question 39

Describe the development of Neural Crest cells

Answer 39

The neural crest is derived from the neural plate, which is induced by Wnt, BMP, FGF morphogens. More specifically, the neural crest cells are derived from the neural plate border cells. The genes that up-regulate ‘neural plate border specifier genes’, include Msx1/2, Pax3/7 & Zic.
• Then upregulate NC specifier genes: including Snail, FoxD3
• Finally, express genes that make them migratory and multipotent.
• An extra-layer of complexity is epigenetics, which can effect these cells.

Question 40

The neural crest cells migrate to form what segments?

Answer 40

There are four main different segments of the neuraxis: cranial, cardiac, vagal, and trunk:
• Cranial NC produces majority of the bone and cartilage of head and face & nerve ganglia, smooth muscle, connective tissue and pigment cells
• Cardiac NC contributes to heart development by forming the aorticopulmonary septum and conotruncal cushions
• Vagal NC gives rise to enteric ganglia of the gut
• Trunk NC gives rise to neurons and glia, & some PNS, to secretory cells of the endocrine system & to skin pigment cells

Question 41

How do neural crest cells migrate?

Answer 41

The neural crest cells migrate along stereotypic pathways (semi-defined) in embryo.
• A dorsal lateral pathway between the epidermis and mesoderm
• And a ventromedial pathway through the somites
This is achieved by attraction and repulsion extracellular signals.

They divide and integrate signals (to know where to stop and what to become) as they migrate.

Question 42

How do neural crest cells transition from migratory to differentating state?

Answer 42

Once they arrive at their determined location, they down-regulate migration genes e.g. Snail. At the same time, lineage switch genes expressed/upregulated.

The neural crest cells become restricted in their fate. As multipotency is lost, and their fate is therefore restricted. The range of fates originally available to them is still very wide, as they subjected to transcription factors from all around the body.

Question 43

Give examples of conditions caused by defects in neural crest cell

Answer 43

• Birth defects: cleft palate, septal defects of the heart and outflow tract
• Hirschprung’s disease (1/5000 live births, congenital absence of enteric ganglia in distal part of colon)

Question 44

Why study the cerebellum?

Answer 44

Why study the cerebellum (Cb)?
• Human Cb develops early embryo – first few postnatal years & is vulnerable
• Human Cb damage and disorders:
○ Ataxias
○ Tumours (e.g. medulloblastoma)
o Cognitive defects (?)Starting to understand functions of its microcircuits

Question 45

What are the functions of the cerebellum?

Answer 45

The cerebellum is involved in motor behaviour. We continuously sense posture, balance & co-ordination and generate pattern of muscle changes etc. for movement. We need the cerebellum to learn how to move in new situations (including timing & sequence).

The cerebellum judges error between intended and actual voluntary movement and we learn from this.

Does the cerebellum have higher cognitive functions?
• Cerebellar cognitive affective syndrome (CCAS) includes impairments in executive, visual-spatial, & linguistic abilities (sometimes without motor symptoms)
• fMRI, PET etc. of Cb lesions & non-lesioned - Suggest Cb activated in language processing (including fluency and word meaning) (attention, working memory, executive functions)

Question 46

Describe the circuitry of the cerebellar cortex

Answer 46

The cerebellar cortex is attractive to developmental biologists because of its patterns and folds. There are three layers:
• Molecular Layer
• Purkinje layer
• Granular layer
Compared to the cortex , the circuitry is quite simple.

Sensory input is fed into the granule cell layer, from which a granule cell feeds a parallel fibre into the molecular layer. This feeds into interneurones to modulate the signals. The one output of the cerebellum is the inhibitory output from the Purkinje cell layer.

Question 47

What are the steps in cerebellar cortex develeopment?

Answer 47

1. Establish cerebellar ‘field’ in the hindbrain. The isthmic organiser secretes signals to define what is hindbrain and what is midbrain. This is needed for the generation of the cerebellum.
2. Form two compartments of cell proliferation: produce Purkinje cells & granule cells
3. Migration of cells
4. Formation of cerebellar circuitry & further differentiation

Question 48

Describe the formation of the two compartments of cell proliferation in cerebellar developement

Answer 48

Step 2: Form two regions of cell proliferation

Zone 1 = rhombic lip (RL), posterior edge of cerebellum field/’anlage’ express Math1+ (a bHLH tf): Produces glutamatergic neurons inc. Deep Cerebellar Neurones (DCNs) - not seen in picture on left, and Granule Cells (GCs).

Zone 2: ventricular zone lines 4th ventricle, Ptf1A+ produces GABAergic interneurons, Purkinje cells and all cerebellar glia.

Question 49

Describe cell migration in cerebellar development

Answer 49

Step 3: Different cell types migrate out of the two zones differently
• Post-mitotic cells from ventricular zone including Purkinje cells - most migrate radially & stop in layers of the cerebellar cortex.
• Cells exit that exist the Rhombic Lip first migrate over the surface of the cerebellum and then move inwards. Deep cerebellar neurones (DCN) neurons leave the Rhombic Lip first & descend ventrally to form 3 pairs of deep nuclei
• Granule cell precursors migrate to the surface of the cerebellum, and form a proliferative secondary precursor zone - external granule cell layer (EGL).

Post mitotic GCs leave the EGL E18.5-P16 and migrate down Bergmann glial cells past Purkinje cell layer to form the IGL (internal granule layer). The granule cells keep their axons (parallel fibres) above the purkinje cells, in the molecular layer.

Question 50

How is cell migration in cerebellar development conducted?

Answer 50

Granule Cell migration is guided through growth cone interactions with neurotrophic factors.
• E.g. netrin receptor (UNC5H3) mutant – GCs migrate into midbrain and brainstem
• Glial guidance cues (weaver mutant) adhesive cues their final destination has ‘stop’ signals

Example of a GC migration regulator
• Trio, a guanine nucleotide exchange factor
• No GCs in IGL and mice are ataxic
• GCs can’t migrate well and have abnormal dendrites (cytoskeleton regulation)

Question 51

What are the role of Bergmann glial cells in cerebellar development?

Answer 51

After Bergmann glial cells are used for migration, they differentiate into unipolar astrocytes with their cell bodies close to Purkinje cells, radial fibres wrap synapses. Purkinje cells and Bergmann glia mature together and presumably ‘talk’ to one another. (Saab et al., Science 2012) shows how Bergmann glia are required for fine motor coordination.
• Glutamate from climbing fibres and GC terminals activate AMPA receptors subtypes on Bergmann glia
• Knock out these receptors in young and adult mice:
○ BG retract, fewer PF:PC synapses, don’t clear the glutamate
When Bergmann glia lacked AMPA recs, eyelid control and ladder stepping declined