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Question 1

Early neuroepithelial cells…..

Name changes to…..

Answer 1

Symmetrical division and give identical neuroepithelial cells.

Asymmetrical division causes a shake change and they are known as radial glia.

Question 2

Following neuron migration

Answer 2

Triated H3 thymidine is radioactive and can incorporate into newly synthesised DNA.

Inject into pregnant female and it will be incorporated into cells in S phase.

Only cells in their final division will keep the H3 label. This will birth date these cells and allow us to trace their migration

Question 3

Layers of the cortex migration basic

Answer 3

Neurons born at different times migrate to different layers of the cortex.

The cells born first occupy the deepest layer And migrate the least.

Inside out development.

Question 4

Precursors plasticity over time experiment.

Answer 4

Heterochronic transplant of early precursors into an older host shows that the early precursors migrate to and adopt to the fate of the cells being born at that time in the host and this means early precursors fate is still plastic.

Later born precursors put into an younger host shows the late precursors migrate and adopt to the fate that they would have if they had not been transplanted. So their fates have become fixed over time.

Question 5

Mutations affecting migration

Answer 5

Cause lissencephaly- smooth brain with no gyri or sulci

Most neurons are found in the deeper layers because they couldn’t migrate away.

The mutations are in genes that code for microtubule proteins.
TUBA1A alpha Tubulin. TUBB2B beta tubulin LIS1 and DCX

microtubules are critical for migration of precursors up the radial glia.

Question 6

Layers of the cortex and how they are made mess.

Answer 6

The first set of neurons leave the ventricular zone and migrate radially to form the preplate.

The second set of neurons migrates radially and passes the preplate. And becomes the first layer of the cortical plate.

The preplate splits into two sections. The sub plate at the bottom and the marginal zone at the top which contains CR cells.

The cortical plate forms between the two separated layers of preplate.

MZ
CR
SB- die
VZ

Question 7

CR cells

Answer 7

First post mitotic cells to appear and become the outermost layer of the cortex (marginal zone)

They change shape and die in the post natal period.

They make sure that the cortical plate layers are ordered correctly.

Question 8

How can we see CR cells

Answer 8

Silver staining

Genetically labelling GFP.

Question 9

Reeler mutant mouse

Answer 9

the reelin gene encodes a large ECM protein that is expressed by CR cells.

Loss of reelin leads to failure of CR cells. The preplate doesn’t separate.
The cortex layers develop in reverse.
The cortical plate forms below the sub plate instead of above it.

The migrating neurons fail to stop.

Causes lissencephaly and ataxia.

Question 10

Radial glia in adult

Answer 10

Some radial glia survive in the adult and become astrocyte like.

Remain in the ventricular zone to become stem cells.

Found in the SVZ of the fourth ventricle and the hippocampus.

Question 11

Tangential migrations

Answer 11

Migration along the layers. Not up and down.

Happens after radial migration and allows the neuron to travel to where they need to be in the adult.

Question 12

Output neurons are developed…

Inhibitory interneurons are not developed…..

Answer 12

In the cortex.

Not developed in the ventricular zone in the cortex.
They tangentially migrate into the cortex from the subpallium.

The subpallium is the source of many other inhibitory neurons.
GABA, dopmainergic to the olfactory bulb, cholinergic to the striatum.

Question 13

Method to trace migration

Answer 13

Transplantation between quail and chick. Their neurons are easy to differentiate between so can be traced.
Or dye the implanted neurons

Injection of viruses into rodents which will infect their ventricular zone. Use fluorescence to track the infected cells.

Question 14

Cerebellum structure. 5

Answer 14

Cortical region and central deep nuclei

The purkinje cells are the main output neurons which synapse onto granular neurons and their axons stretch into the molecular layer.

The cerebellum forms at the roof of the fourth ventricle from specialised roof plate cells at the journey of the midbrain and the hind brain.

Rhombus kite shaped.

Has both radial and tangential migration

Question 15

Roof plate cells that don’t migrate away near the cerebellum

Answer 15

Rhombic lip cells and they become cerebellum neurons.

The anterior rhombic lip becomes granule neuron precursors and the posterior rhombic lip becomes the pontine nuclei.

Question 16

Granular neuron birth

Answer 16

The dark like around the developing cerebellum is the external germinal layer
It disappears in the adult.

The neurons are born outside the cerebellum and migrate in.

The neurons migrate tangentially and then extend their cell body radially into the internal granular layer.

They extend axons away from their migrating cell body.

They then form synapses with the purkinje cells.

Question 17

Purkinje cell birth

Answer 17

Born in the normal way at the ventricular zone.

Question 18

Factors controlling cerebellum development

Answer 18

Production of rhombic lip cells is regulated by MATH1
No MATH1 means no formation of the pontine nuclei and inferior olive.

Pontocerebellar hypoplasia- fewer cells in pons and cerebellum. MATH1 mutation.

The pons and external germinal layer must have similar origins.

Question 19

Levels of shh and cerebellum

Answer 19

Shh is released from purkinje cells and stimulates mitosis on the external granular layer. This makes granule neurons form.

Affect the amount of lobulation and proliferation

Too much shh leads to medulloblastoma in children because the granule neurons proliferate our of control.

Question 20

Filapodia and lamellapodia

Actin

Answer 20

Reach out and explore the environment

Fila are fingers and on microscope they are strings. Lamella are webs.

Peripheral is tips.
Transitional is bottom of finger.
Central is cell body.

Lamella- the F actin bundles are cross linked into a net or basket.
Fila- the actin bundles are polarised to form larger bundles. Linear.
They are both highly motile.

Question 21

Aplysia

Answer 21

Used to study growth cones because theirs are very flat and you can see what’s going on inside.

Question 22

F actin tread mills in a resting growth cone

Answer 22

No obvious movement in central domain.
In peripheral domain, the F actin fibres are flowing in towards the central domain.

In the transitional domain the filaments start to break up and the F actin gets broken down into actin.
Actin is being added to the Fila tips which joins the F actin chain. It then flows down to the centre and is broken back into actin.

Tubulin is dragged from the central domain and is shooting up the backs of the Fila to the tips.

Question 23

F actin tread mills in active growth cone.

Answer 23

Happens dramatically

Eg a polystyrene bead soaked in an attractive cue will activate it.

F actin treadmilling slows and this causes F actin accumulation. Less is being broken down to actin.
This will stabilise the Fila and drag lots of tubulin into the back of them.

If the bead was immobile the growth cone would reorganise itself to establish a new direction.
They don’t turn they search the environment for a cue and then flood their cytoskeleton with F actin and Tubulin so it can be reorganised to grown in another direction

Question 24

What two things happen after a promoting cue is encountered

Answer 24

A molecular clutch in the Fila is engaged. Slowing down the central flow of F actin.
Actin subunits are still being added to the tip

The second this is that an actomyosin based actin-Tubulin link pulls microtubules of Tubulin into the wake of the extending Fila.

Question 25

What two things are both needed for growth cone movement

Answer 25

Attachment of substrate Stimulus of a cue/ allowing growth Both permissive and non permissive substrates together guide the axon.

Question 26

Repulsive cues discovery and what happens.

Answer 26

Mixtures of neurons in culture were found to fasciculte with their own kind. This was due to different neurons repulsing each others axons. After the encounter the growth cones collapse and new growth cones are made that point in the opposite direction. It destabilises F actin and causes the Fila to have very low levels of it. And the cytoskeleton will collapse and move away.

Question 27

Semaphorins 4 What they do and knockout

Answer 27

Family of inhibitory guidance cues. Come in many flavours Can be membrane bound or secreted. Cause growth cone collapse. Semaphorins channel the T1 axon to cx1. It’s function can be blocked by antibodies. Mice lacking semaphorin 3a have axons straying into the wrong territories.

Question 28

Where do axons prefer to grow

Answer 28

Sticky areas (polyornithine) As long as they can stick it doesn’t matter how sticky it is. Collagen is more sticky than laminin. But laminin is favoured by axons to grow on. So it is not that the more sticky the surface the more axons grow there.

Question 29

What type of factor is laminin and why

Answer 29

It is a growth promoting ECM protein and is localised in the optic nerve. It does not direct the direction of growth. Just that they can grow. So it a permissive factor and not directional/instructive Blockade of the receptors for laminin slows down the growth of retinal axons by does not change their direction. Laminin is only permissive within a certain concentration range. Too much or too little means no growth.

Question 30

Permissive substrates Non permissive substrates

Answer 30

Contact attractants Contact repellents.

Question 31

What causes growth come collapse

Answer 31

Not due to lack of attachment because it is still attached when it collapses. It’s the signalling.

Question 32

Ephrins

Answer 32

Non permissive contact repulsion factors on the cell surface detected by eph receptors on axons. They are used to compartmentalise the embryo because of their reciprocal pattern of expression. They cause repulsion between cells to help separate rhombomeres. And they keep axons out of specific areas.

Question 33

Chemo attractants and repellants in the commisural axons

Answer 33

Tell axons which direction to grow. Commisural sensory relay neurons grow towards the floor plate because it is secreting netrin. Netrin has a similar structure to laminin and can associate with the ECM. The axons are also repelled by BMP made in the roof plate. It causes growth cone collapse.

Question 34

Molecules that are used early on to pattern where specific neurons are born also...

Answer 34

Guide axons later on

Question 35

Netrin knockout mice.

Answer 35

Some commisural axons are still able to reach the floor plate. The normal path of these axons is to then cross the midline. And none of them did this. This could be because the shh in the floor plate guides the axons. if you add cyclopamine this blocks shh and the neurons can’t reach the floor plate anymore. Smo knockout also leads to disruption to axon pathway.

Question 36

Cre recombinase and what it can make

Answer 36

Bacteriophage P1 encodes cre recombinase and allows it to insert its DNA into the host bacteria’s genome. Cre binds to a specific 34 base sequence called loxp. Which it can cut and rejoin to another loxp site. We can use this to delete DNA lying between two loxp sites. A floxed gene is one that’s surrounded by loxp sites. And can be deleted using cre recombinase A stop codon could be deleted to allows activation of other genes. Can create smo knockout only in commisural axons or it would be early embryonic lethal.

Question 37

Shh and netrin work together to.. Shh and BMP are used early for... And late for ...

Answer 37

Guide axons to the floor plate Early to specify neural fate Late to guide commisural axons.

Question 38

Long and short range cues interacting.

Answer 38

Semaphorin 1 is on the cell surface. Semaphorin 2 is secreted. They work together to guide the T1 axon. 1 is a short range cue 2 is long range. You need both for axon guidance to be successful.

Question 39

Contact attraction example

Answer 39

Cadherins

Question 40

Axon reprogramming and example

Answer 40

They reprogram when choice points are encountered. Commisural axons lose response to netrins after crossing the midline. So they are no longer attracted to the floor plate and can pass it and ascend upwards.

Question 41

Open book experiment

Answer 41

The neural tube is cut along the roof plate and resembles an open book. The floor plate is in the middle and the two halves of the roof plate are at either end. Lipophilic dye is added. Highlights cell membrane and are absorbed because they are lipophilic. Shows axons and dendrites. Show that axons are only attracted to the floor plate before they cross the midline. After crossing they change their sensitivity to netrin and are no longer attracted to it.

Question 42

After crossing the midline... Commisural axons

Answer 42

Axons don’t respond to netrin. They now respond to repellants. Commisural axons grow parallel to the floor plate after passing it. This is because of the inhibitory molecules in the floor plate and ventral spinal cord that creates a channel where the axons can grow. They avoid the semaphorins and slits that are in the floor plate and ventral spinal cord. Initially the axons ignore the repellants and are attracted to netrin before they cross the midline.

Question 43

Hindbrain axons crossing the midline.

Answer 43

They ascend away from the floor plate after crossing the midline.

Question 44

Drosophila 3 facts about axons.

Answer 44

The midline is conserved in vertebrates and flys. Drosophila have netrins and slits too. They both have axons crossing the midline.

Question 45

Roundabout gene

Answer 45

Mutation causes axons to cross the midline multiple times and don’t form longitudinals. Robo encodes slit receptor. Commissural axons express low robo so they are able to cross the midline. After they cross they express high levels of robo, meaning lots of slit and more axon repulsion to stay longitudinal. Mutation means theres no slit. So nothing causes repulsion of axons. So they continue to be attracted to the floor plate and can cross many times creating the roundabout phenotype.

Question 46

Commisurless gene

Answer 46

Mutation is no crossing of the midline. It will normally allow crossing of the midline. And it is only expressed in commisural axons because of this. It is switched off after crossing so that the axon only crosses once. Comm mutants have high robo expression everywhere. Meaning lots of slit and lots of axon repulsion and no crossing over. If comm expression is forced in all axons they keep crossing the floor plate and there is no robo made to repress it. This will give a phenotype like the mutant robo.

Question 47

Comm and robo link

Answer 47

Comm encodes a trafficking protein that prevents robo protein from reaching the cell surface. This means the growth cone won’t recieve slit signals. After the axon has crossed over Comm expression stops. So robo can reach the cell surface and cause slit signals which will repress the axon crossing the midline again.

Question 48

Comm and robo vertebrate homologues

Answer 48

No vertebrate homologues of comm Robo1 is expressed on commisural axons before and after crossing. Robo3 is only expressed before crossing and its job its to block robo1 Robo3 stops robo1 from expressing slit before the axon crosses. After crossing robo3 turns off and robo1 can express slit and prevent the axon crossing again. Loss of robo3 means axons can’t reach the floor plate.

Question 49

Axon fascicultion

Answer 49

Follower axons homophillically bind by (CAMs) cell adhesion molecules such as fas2. If fas2 is expressed in cells that do not normally adhere it causes aggregation. Fas2 controls fasciculation in flies and a mutation causes many defasciculated axons. Over expression leads to bundling of axons that would not normally do so.

Question 50

Drosophila motor neurons and fas2.

Answer 50

There are two different types of motor neuron, RP1 and RP3. They come from the CNS and travel to a muscle block and innervate it. Over expression of fas2 causes a bypass phenotype where the motor axons fail to defasciculate from the bundle and miss their target muscles.

Question 51

BEAT protein

Answer 51

Interferes with CAM mediated adhesion. Causes defasciculation and the axons can reach their targets.

Question 52

What will motor axons do if their target muscle is ablated.

Answer 52

The axon won’t defasciculate and will carry on past the gap when it should leave to join with the muscle. This suggests the growth cones are searching for their target muscles specifically. DISCRETE TARGETS

Question 53

Netrins in muscles.

Answer 53

They are expressed in muscles to signal to the growth cones to find their correct muscle. Loss of netrin is the same as ablating the target muscle. Ectopic netrin in the wrong muscle causes the axon to innervate the wrong muscle.

Question 54

Fas3

Answer 54

Is expressed in muscles and the motor axons that innervate them. Ectopic fas3 causes the fas3 expressing axons to innervate new targets.

Question 55

Maintaining topology by neighbouring neurons sending axons to neighbouring sites Two theories

Answer 55

Sperry 1- each axon has a unique label complementary to a unique label on the target. However this would require many genes to code for each label. 2- coordinate system. Encoded for by gradients of signalling molecules. It stamps a latitude and longitude onto cells of the target which is read by complementary gradients of receptors.

Question 56

Stripe assay

Answer 56

Strips of the tectum membrane were cut out and set up in alternating anterior and posterior strips. Nasal axons normally extend into the posterior of the tectum. They are able to grow on both the A and P slices. Temporal axons could only grow on the A sliced. The temporal axons are avoiding a repellant of two ephrins A2 and A5 in the posterior tectum. There is an ephrin gradient in the tectum, higher conch are in the posterior There is an eph receptor gradient in the eye. With high concs being in temporal side of the eye. If there are more ephs in the temporal side. Then their axons will detect ephrins more often and be repelled from the high conc of ephrins in the posterior tectum.

Question 57

Mice with ephrin A2 and A5 Knock out.

Answer 57

Temporal neurons can grow into the posterior tectum. Because there are no ephrins to repel them. The topographic map is distorted.

Question 58

Crossed retinal ganglia

Answer 58

Stereo vision- light from one object will reach both eyes. Ephrins are used whether a organism has it or not.

Question 59

Targets are selected by :

Answer 59

Address labels on discrete cellular targets. The labels are likely to constitute combinations of molecules. Gradients of ephrins on multicellular targets where topographic maps are to be formed.

Question 60

Trophic vs tropic

Answer 60

Trophic means food Tropic means turning.