Week 3

Question 1

How do we go from a simple neural tube to an elaborate brain?

Answer 1

The tube is made of neuroepithelial cells

This is a single layer of cells; rapidly dividing ‘stem cells’

At this stage it is a single layer of cells

Made up of neuroepithelial cells - make neurons

Looks like lots of nuclei arranged in multiple rows but no.
Pseudostratified layer - nuclei arranged in different places in the single layer of cell. Not random
Where the nucleus is positioned shows position in cell cycle

Position of the nucleus indicates what point of the cell cycle has been reached

Question 2

What happens when you want to make more than just neuroepithelial cells?

Answer 2

Neuroepithelial cells are progenitors for both the neurons and glia

To generate neuron and glial cell types, the neuroepithelial cells change plane of cell division

2 new cells, one stays at ventral zone and keeps cycling, the other (the neuroblast) migrates away (asymmetric division)

Question 3

What do we know about progenitor cells?

Answer 3

They can make more than one type of cells

For this we’ll focus on neurons and glia

To make the specific cells either neurons or glia, they have to change the plane of cell division

Question 4

What does symmetric division of progenitor cells produce?

Answer 4

More of the neuroepithelial cells

Question 5

What does asymmetric division of progenitor cells produce?

Answer 5

The mitotic spindle rotates and the cell splits and one maintains contact with the luminal surface (apical) and one migrates away towards the basal surface, now has the potential to go off and differentiate (post mitotic) during the brain and spinal cord formation

Question 6

What happens in symmetric division

Answer 6

There is verticle cleavage of the progenitor cells

Produces neuroepithelial cells

Question 7

What happens in asymmetric division?

Answer 7

Horizontal cleavage

generates neuroblasts

Question 8

What do we know about the formation of layers/zones in the spinal cord?

Answer 8

The newly formed neuroblast migrates to form a new layer - the intermediate layer (mantle layer)

Cells differentiate and the axons extend into the marginal zone, become myelinated and form white matter

Intermediate remains grey matter (cell bodies)

Question 9

What happens in the cerebrum that is different?

Answer 9

The ventricular zone expands to form a subventricular zone. Together these make the “germinal strata”

Then there is the intermediate zone

Cortical plate (lots of cell bodies) (neocortex)

Finally, marginal zone

Cerebral cortex split into ventricular and sub-ventricular zone, intermediate zone, cortical plate and marginal zone

Sub-ventricular zone - the cells begin to move away from the intermediate zone which is the white matter. Mainly axons, a few cell bodies. The axons are in different directions
Cortical plate - neocortex - this is lots of cell bodies, grey matter
Marginal zone - molecular layer - some white matter but not many cell bodies

Question 10

What is the Germinal Strata?

Answer 10

The ventricular and subventricular zones

Generates radial glial cells, short neuronal precursors and intermediate progenitor cells

Neuroepithelial cells in germinal strata called radial glial cells

Also different cells like short neuronal precursors, give rise to cells which migrate away

Also intermediate progenitor cells - arise from radial glial cells and then differentiate and give rise to other cell types

Radial glial cells dominant cell type in ventricular zone
Intermediate progenitor cells only in sub-ventricular zone

Difficult to see as two specific different zone through a microscope but can be seen on a molecular level

Cux2, Tbr2, Satb2, Nex, Svet1 all transcription factors in Sub-ventricular zone
Otx1, Fez1 transcription factors in Ventricular zone.
Turn on genes for cell production, ensure the right cells develop in the right place

Question 11

What is radial glia?

Answer 11

Radial Glia span the width of the developing cerebrum

Are now thought to be “stem cells”

Also act as important scaffold for neuroblast cells to migrate along

The cells primarily migrate into the cortical plate

The majority of cells originate from radial glial cells

Question 12

What is the role of radial glia?

Answer 12

Two important jobs, proliferate and produce new cell types and act as scaffolding within the developing brain.

Migrating cells post asymmetric mitosis will use RGC as a scaffold/rope to climb up as it migrates away to the basal surface.

Interesting mechanism, not seen very often, not only creates the cell but acts as a scaffold to guide it to the right place

Question 13

What cell signalling helps the radioglial cells?

Answer 13

Delta-notch signalling very important, two cells next to each other can communicate across membranes to coordinate activity

Mix of transcription factors and signalling molecules involved to ensure migrating cells move and end up in the right place

Get rid of some of these and the migrating cells either dont move or end in the wrong place

Question 14

What do we know about radial glia and cell division?

Answer 14

The RGC must shuttle the nucleus from the basal to apical (ventricular) surface to divide

This is called interkinetic nuclear migration

Controlled by many transcription factors

Disruption of these factors reduces the production of RGCs and therefore progenitor pool

E.g. Sun1 and 2 knockout mice have smaller cortices because they have fewer Radioglial cells

Zika virus makes cortex small - microcephaly - viruses can interrupt the signalling in the shuttling process

Shuttling process very disruptable

Question 15

What do we know about intermediate progenitor cells?

Answer 15

Two subpopulations - the apical intermediate progenitor cells (aIPs) and basal Ips (bIPs) - distinct molecular profiles

aIPs reside in the Ventricular zone (VZ). Short radial attachments to the apical (ventricular) surface

bIPs delaminate from the VZ and migrate into the SVZ

IPs migrate into the cortex and usually divide symmetrically to generate two postmitotic neurons

Question 16

What do we know about the process of radioglial cells giving rise to new cells?

Answer 16

We know most about this process in developing mice but think its almost identical in humans

E12 and E14 - embryonic day 12 and 14

Not happening at the very early stages of development

Day 12 RG cells proliferating

After E14, RGC proliferating and making new RGC, anything being produced is now basal intermediate progenitor cell which migrates into SVZ for a short amount of time, like rest

Then moves into the mature cortex and divides to produce two neurons

aIPs migrate and dont proliferate further

bIPs migrate and then proliferates - unusual for neurons, make them and then post-mitotic and then migrate further into layers II/III. These migrate and proliferate. Neat way of making sure you have plenty of neurons. A lot more flexibility than aIPs

Question 17

What do we know about migration out of the ventricular zone/subventricular zone?

Answer 17

Cajal Retzius pioneer neurons migrate out of the VZ/SVZ and generate a transient layer called the preplate

Further migration of other neurons creates the splitting of preplate into subplate and Cajal Retzius layer (also called Marginal layer)

The Cajal Retzius cells end up in the molecular layer (Layer I of cortex)

Other 5 layers of the neocortex form below this

Pre-plate region migrate out of VZ, SVZ and then Pre-plate - first lot of cells

These cells are a group of pioneer cells which managed to migrate away without the RGC scaffold - Cajal-retzius cells

Create the Cajal-retzius layer

Act as a barrier on the outer layer of developing brain (mature brain molecular layer)
All other layers form behind this layer

Question 18

What do we know about the formation of the cortical layers?

Answer 18

The subplate and the marginal zone/CR layer become the edges of the cortex and new layers are sandwiched in between (layers 2-5)

Question 19

What do the radial glial cells do to the Cajal Retzius cells?

Answer 19

They extend the scaffold up, allowing the cells to migrate all the way up

E16.5 lots of different layers after cell migration
- Marginal zone - Layer 1 - Cajal-Retzius
- UL - Layers 2-4
- DL - Layers 5-6
- Subplate
- Intermediate zone
- Subventricular zone
- Ventricular zone

Question 20

What do we know about the “inside out” formation of layers?

Answer 20

The first cells migrating from the VZ/SVZ end up in layers 6 and 5

Subsequence waves of neurons end up in layers 4, 3, 2 in that order

Thus, the neurons born post-natally end up in the outermost layers of the cortex

The precise control of this ordered migration is critical for cortical architecture

Highly dependent on radial glial cells

Question 21

What neurons are in which layers of the cortical plate/neocortex?

Answer 21

The area of the cell body is where the cell differentiated and the axon then extends to other parts of the brain

Layer 2/3 - Other cortical areas
Layer 4 - axons from thalamus
Layer 5 - Corpus callosum and spinal cord, superior colliculus, pons
Layer 6 - Thalamus

Question 22

How does the formation of layers in the cortical plate/neocortex occur?

Answer 22

First, cells that migrate move to the closest layers

Then, the next cells squeeze past cells in the closest layer to the next layer

And this continues until the outermost layer cells have migrates

Tightly controlled process involving hundreds of genes over a vast period of time

RGC necessary as we need the production and scaffold

Humans still growing brains up until around 2 years

The layer formations are happening post-natally

Question 23

What do we know about Layer I - the molecular layer - of the cortical plate?

Answer 23

First cortical layer to form

Cajal Retzius Neurons migrate to this layer without using radial glial cells

May be aided by meningeal cells

They produce a protein called REELIN

This aids neuronal cells in VZ/SVZ to migrate along RGCs and positions them in appropriate layers underneath

Reelin mutants have disorganised cortex

This layer also contains pyramidal cortex cells

Question 24

How do we know the cortex is split into layers?

Answer 24

Gene expression studies

Only molecular layer is visibly different
- Mostly axons
- Cajal-Retzius neurons
- Meningeal layer guides the Cajal-Retzius neurons to form the molecular layer
–> Produce REELIN, chemoattractant which encourages migration of the pioneer cells

Without REELIN, the cortex is hugely disorganised

Question 25

What do we know about Layer II, the external granular layer, of the cortex?

Answer 25

Last layer to form Contains pyramidal neurons, astrocytes, stellate cells and radial glial cells Pyramidal glutaminergic neurons make up 75% of cortex. Remaining 25% are GABAergic interneurons

Question 26

What do we know about Layer III, the external pyramidal layer?

Answer 26

Contains pyramidal neurons, astrocytes, Stellate Cells and radial glial cells

Question 27

What do we know about layers 4-6, the inner granular layer, ganglionic layer and the multiform layer?

Answer 27

Contain Stellate, pyramidal and multiform neurons

Question 28

What is the final phase of neocortex formation?

Answer 28

Gliogenesis. where you make glial cells E10 make loads of RGC E11-E16 making new neurons Post E16 of dev up until after birth RGC largely making glial cells

Question 29

What is tangential migration of GABAergic interneurons?

Answer 29

Calm the system down - inhibitory Without enough GABAergic cells, conditions like epilepsy or schizophrenia occur Majority originate in the ganglionic eminences in ventral region of developing brain - Medial ganglionic eminence cells are important as they produce GABAergic interneurons - GABAergic interneurons migrate at right angles - as a tangent to the developing structure

Question 30

How are gyri formed?

Answer 30

The formation of gyri and sulci allows you to pack more cells into a small space Not all species form gyri These species are naturally lissencephalic, like the rat

Question 31

What is lissencephalic?

Answer 31

Smooth-brained

Question 32

What do we know about Basal Cell Expansion in the VZ/SVZ?

Answer 32

May control Gyri formation Formation of gyri seems dependent on bIP cells and bRGCs Expansion of these populations leads to formation of gyri FGF2 signalling may have a big impact on gyri formation This cell type is why we need folds on our brain

Question 33

Remind us of the development of the cortex?

Answer 33

Mature cortex has 6 layers The first cells exit the ventricular zone (VZ) towards the cortical plate (future cortex) to form the pre-plate (outer Cajal-Retzius neurons, inner subplate neurons) Subsequent cells from the ventricular zone enter between these two layers of the pre-plate in an inside to outside sequence (layers 6 to 1)

Question 34

What does the growth of the cortex rely on?

Answer 34

The expansion of neural stem cells (NSCs) and neural progenitors (NPs) and the subsequent generation of postmitotic neurons

Question 35

What is lissencephaly?

Answer 35

Class of human cerebral malformations characterised by the agyric surface of the brain

Question 36

Based on cortical folding, what can mammals be divided into?

Answer 36

Lissencephalic species (such as mice), which have smooth-surfaced cortices Gyrencephalic species (such as ferrets and most primates), which exhibit convolutions in the cortex

Question 37

What do we know about Lissencephaly?

Answer 37

Severe cases cortex lacks surface folds (agyria) Milder manifestations abnormally broad folds (pachygyria) Heterotopic layer of grey matter embedded in white matter (subcortical band heterotopia SBH) Prevalence 11.7 to 40 cases per million

Question 38

What is pachygyria?

Answer 38

Fewer folds and broader

Question 39

Why is lissencephaly important?

Answer 39

Understanding the process of cortical folding in the mammalian brain has direct implications on the diagnostics of neurological disorders including severe retardation, epilepsy, schizophrenia and autism

Question 40

What are the types of Lissencephaly?

Answer 40

Type I Lissencephaly - "Classical Lissencephaly" lissencephalies associated with a failure of normal cell migration Type II Lissencephaly - "Cobblestone lissencephaly" lissencephalies associated with apparent over migration of neurons

Question 41

What is Type I Lissencephaly?

Answer 41

Characterised by an abnormally thick 4 layer cortex and absence of other brain abnormalities Caused by mutations in genes affecting microtubules - PAFAH1B1 (Lis1) - DCX - TUBA1A Mutations affect cell migration, proliferation and differentiation, i.e. neurite outgrowth, axonal pathfinding, axonal transport, connectivity and even myelination

Question 42

What do we know about PADAH1B1 (Lis1)?

Answer 42

Encodes for a highly conserved 45kDa protein Key role in cell division and cell motility mediated by binding to other proteins Able to bind to and regulate cytoplasmic dynein Required for movement during neuronal migration Associated with a range of severity no clear genotype/phenotype correlation

Question 43

What do we know about DCX?

Answer 43

Also known as XLIS, enriched at the extremities of neuronal processes Lyonisation (X chromosome inactivation) produces two populations of cells in female heterozygotes - neuroblasts with the DCX allele inactivated undermigrate and form the heterotopic band In hemizygous males on the defective DCX allele will be active leading to a more severe phenotype DCX mutations cause a classical lissencephaly with an A>P gradient

Question 44

What do we know about TUBA1A and other tubulinopathies?

Answer 44

Microtubules play an important role at multiple stages of cortical development - Pushing and pulling forces during migrations - Generation of and stabilisation of axonal processes - Synaptogenesis - P>A gradient - Mutations also found in TUBB2B and TUBB3 genes associated with polymicrogyria-like cortical dysplasia - Mutations in TUBA8 - polymicrogyria - Mutations in TUBG1 - pachygyria

Question 45

What do we know about Type II lissencephaly?

Answer 45

Distinct from classical lissencephaly, can appear agyric or pachygyric Cortex typically thinner than in type 1 Due to overmigration of neurons through breaches in the pial basement membrane Their accumulation in the sub-arachnoid space giving the cortex an uneven surface Mostly due to defects in the glycosylation of alpha-dystroglycan

Question 46

What are cerebellar and ocular abnormalities and congenital muscular dystrophy associated with type II lissencephaly?

Answer 46

Walker-Warburg Syndrome (WWS) - hydrocephalus, agyria, retinal dysplasia, muscular dystrophy - muscle-Eye-Brain disease (MEB) Perhaps most common syndrome Genes involved in type II have major role in muscles Fukuyama congenital muscular dystrophy (FCMD) Congenital muscular dystrophy type 1D (MDC1D) All involve defect in O-mannosylation (glycosylation disorders) Post translational modification of a protein Defect in how mannose is added to those proteins

Question 47

What is dystroglycan?

Answer 47

Non integrin adhesion molecule first identified in skeletal muscle and brain Comprised of two subunits, alpha and beta, the alpha subunit is extensively glycosylated Dystrophin part of muscle development This process also part of brain development

Question 48

What happens to alpha-dystroglycan?

Answer 48

Glycosylation - allows things to attach to the alpha-dystroglycan. If you lose those points all the things that attach to and signal through alpha-dystroglycan cannot attach to it

Question 49

What happens to the basement membrane in cobblestone lissencephaly?

Answer 49

It is abnormal Holes in basement membrane, lose some neurons as they escape and create bumps on the surface of the brain Alpha-dystroglycan works to make the basement membraine work properly

Question 50

What do we know about X-linked lissencephaly with ambiguous genitalia?

Answer 50

Mortality in first year high Caused by mutations in the ARX gene (Aristaless-related homeobox protein) ARX known to regulate genes involved in cell migration, axonal guidance, neurogenesis Associated with a wide range of diease phenotypes, including non-syndromic learning disability ARX-related lissencephaly shows three cortical layers, primarily due to aberrant tangenital migration of GABAergic interneurons into the cortical plate 3 cortical layers instead of 6 GABAergic migration problems

Question 51

What do we know about Baraitser-Winter Syndrome?

Answer 51

Very Very Rare Syndrome Characteristoc facial features Anterior predominant pachygyria Intellectual disability and epilepsy - severity correlates with degree of malformation Linked to gain of function missense mutations in ACTB and ACTG1, other phenotypes also linked to mutations in these genes

Question 52

What do we know about lissencephaly with cerebellar hypoplasia?

Answer 52

Approx. a third are due to mutations in TUBA1A Also associated with mutations in RELN - A>P gradient - Severe abnormalities of cerebellum, brainstem, and hippocampus Mice with homozygous mutations (REELER mice) have inverted cortical layering (later born neurons are deeper to early-born neurons) and cerebellar hypoplasia RELN encodes a large extracellular glycoprotein (around 400kDa) Expressed by Cajal-Retzius cells in the marginal zone where it acts as a 'stop' signal for neurons migrating into the cortical plate RELN - glycoprotein Stop signal Tracks originally and then stops Loss = completely disorganised cell migration

Question 53

What is microlissencephaly?

Answer 53

Associated with a head circumference at birth of less than 3 SD below the mean Homozygous mutations in the NDE1 (nuclear distribution factor E-homolog 1) gene underlying cause Profound cognitive impairment, brain atrophy and extreme microcephaly (>10 SD below the mean) Protein encoded by NDE1 localises to the centrosome and mitotic spindle poles Gene NDE1 - nuclear distribution factor E homolog 1 - Mutations main underlying cause NDE1 one of main roles makes mitotic spindle - possibly not reorientating for symmetric and assymetric division so brain possibly doesn’t fold due to lack of neuron cells produced so plenty of space

Question 54

What are the phenotypes of the cortical abnormalities in the presence of specific gene mutations?

Answer 54

ARX - Quite severe phenotype 3 layers TUBA1A - Very severe phenotype Only 2 layers The fewer layers you have the more cognitive defects

Question 55

What is the Brainbow mouse?

Answer 55

A multi-gene and exome sequencing. Insertion of fluorescence genes from coral, jellyfish and bacteria into the mouse genome making mice brains shine in an array of colours illustrating a range of complex neuronal connections that would not be evident using more conventional stains Cells labelled in more than 100 different hues enabling a comprehensive map of neuronal connections to be built up. Can now look at whole genomes, all mRNA expressed in a single cell at any one time - Enormous power to see how different cell types are defined etc Standard lab can now do whole genome sequencing, mRNA sequencing etc Becoming cheaper each year Brainbow mouse - nifty tool - insert fluorescent proteins into mouse genome - makes brains light up. Proteins from jellyfish, bacteria etc Can really see the layers Now more than 100 proteins we can insert so you can really see the differences

Question 56

Why are brainbow mice so good?

Answer 56

Much more vibrant Much more complex Much easier to see different cells, axons and how they connect Can see the layers really easily

Question 57

What do we know about brain organoids, spheroids and assembloids?

Answer 57

Advance in tech Ability to make brain organoids aka. Spheroids and Assembloids Can take a cell from skin, induce it to become embryonic and then assemble into embryoid body and culture it to become a brain organoid If you take a cell from someone with a developmental defect and do this you can see where their defect came from Can use human tissue or tissue of species you're most interested in Also helps us know how cells work together (spheroid fusion) but cerebral organoid is where most research is right now