Cortical development and neural diversity

Question 1

Describe the methods of an experiment where they analysed hippocampal proliferation in-vivo

Answer 1

At P60, they did an I.P EdU injection and 24h later extracted the brain and collected coronal slices of the hippocampus.

They then stained the slices with EdU to stain the S phase (DNA replication) and Ki67 (growth factor). Ki67 stains during the cell cycle outside of S phase.

Question 2

What are these stages after the S phase?

Answer 2

G2 (Protein synthesis and checks the duplicated chromosomes for errors)

=> M phase (Mitosis)

=> G0 phase (cell cycle arrest: Normal function/quiescence)

=> G1 (Cell growth: cellular contents excluding the chromosomes are dublicated)

Question 3

What would EdU+, Ki67+ and EdU+Ki67+ NSCs indicate?

Answer 3

EdU+: In S-phase during EdU pulse, but not in cell cycle during sacrifice

Ki67+: NOT in S-phase during EdU pulse, but was in cell cycle during sacrifice

EdU+Ki67+: In S-phase during EdU pulse, and in cell cycle during sacrifice

Question 4

What staining should also be done as a follow up?

Answer 4

Should do another immuno-staining to make sure they are stem cells. If you do this at different time points, the less EdU+ and Ki67+, as there are less stem cells and those which are still there are in deeper quiescence.

Question 5

Name three complexes and checkpoints involved in the cell cycle and when they occur

Answer 5

The cyclin B-Cdc2 complex begins at the initiation of the M phase until the exit of the M phase. There is a checkpoint at the end of the G2 phase and at the exit from the M phase.

The cyclin D-CDK4/6 complex begins halfway through G1 until the initiation of the S phase. The cyclin E-CDK2 complex is activated from halfway through G1 until halfway through the S phase. There is a checkpoint at the initiation of the S phase.

Question 6

What is the purpose of these checkpoints?

Answer 6

After S phase it is checked whether the DNA was replicated properly. During G1 there is a checkpoint for other intracellular components such as proteins to ensure they are split properly. If it is not sufficient there may be cell death or senescence.

Question 7

What is the relationship of CDKs and cyclins?

Answer 7

Cyclins (CCNs) are activators of CDKs (Cyclin dependent kinases)

Question 8

Describe the concentrations of 4 different cyclins over the cell cycle

Answer 8

Some are specific for a phase, others are globally important, there are many different cyclins:

The G1 cyclin forms a gaussian distribution over the entire cycle, beginning at G1, ending at M and peaking over the S phase.

The G1/S cyclin froms a narrow normal distribution spanning halfway through G1 and S phases, peaking halfway through.

The S cyclin slowly builds up from the end of the G1 phase, rising through the S phase and peaking in the G2 phase.

The M cyclin slowly builds up from the beginning of the S phase, rising through the G2 phase and peaking in the M phase.

Question 9

Describe what happens when G1/S is present or not in the cell cycle

Answer 9

When G1/S is absent, CDK is inactive it does not influence its target genes and the S phase factors are inactive.

When G1/S is present it binds to CDK and activates it so it can phosphorylate targets and DNA replication enzymes are activated and the S phase begins.

Question 10

What happens when M cyclin is present during the cell cycle?

Answer 10

When M cyclin is present it binds to CDK and activates it so it can phosphorylate targets. The spindle forms, chromosomes condense and nuclear membrane brakes down. The M phase begins.

Question 11

What happens if DNA is damaged in G1?

Answer 11

p53 will interact with the CDK ihibitor P21 and this will bind to the CDK-Cyclin complex to inactivate it and therefore the cell is paused in G1. A cell will not enter S-phase when the DNA is damaged.

Question 12

What therefore are the master regulators of the cell cycle and what relevance does this have?

Answer 12

Cyclin dependent kinase inhibitors are master regulators of cell cycle. They are chemicals used in cancer therapies (many in clinical trials)

Question 13

Name three Cyclin-CDK inhibitors from one family and four from another

Answer 13

• Cip/Kip family: p21, p27 and p57
• INK4 family: p15, p16, p18 and p19

Question 14

Describe a relevant point each about the members of the Cip/Kip family

Answer 14

• P21: induces DNA damage cell cycle arrest
• P27: upregulated in quiescent cell states
• P57: primarily involved in embryogenesis (generation of adult stem cells in the embryo)

Question 15

What inhibitors inhibit what complex in the M phase?

Answer 15

p21, p27, p57 =| CyclinB-CDK1

Question 16

What inhibitors inhibit what complex at the start of the G1 phase?

Answer 16

p15, p16, p18, p19 =| CyclinD-CDK6 / CyclinD-CDK4

Question 17

What inhibitors inhibit what complex at the end of the G1 phase?

Answer 17

p21, p27, p57 =| CyclinE-CDK2

Question 18

What inhibitors inhibit what complex in the S phase?

Answer 18

p21, p27, p57 =| CyclinA-CDK2

Question 19

What inhibitors inhibit what complex in the G2 phase?

Answer 19

p21, p27, p57 =| CyclinA-CDK1

Question 20

What happens if you knock out or upregulate p21

Answer 20

p21 is very important in maintenance, if you knock it out you lose your stem cells. If you upregulate it more stem cells go into quiescence.

Question 21

How are CDK inhibitors upregulated?

Answer 21

CDK inhibitors are regulated at multiple levels:
* Transcription
* Phosphorylation (translocation)
* Binding partners

Question 22

What are the Cip/Kip family of CDK inhibitors regulated by?

Answer 22

• P21 transcription is regulated by p53 (DNA damage)
• P27 transcription is regulated by FOXO3 (stress)
• P57 transcription is regulated by Notch/Hes1, BMPs (embryogenesis)

Question 23

So CDK inhibitors are tumour supressors?

Answer 23

CDK inhibitors are not simply tumor suppressors, but are involved in an array of
processes, e.a. apoptosis, transcription and migration

Question 24

How does cell cycle affect NSC fate decisions? (2)

Answer 24

Two main hypotheses on neural fate determination by cell cycle

cell cycle hypothesis
* G1 length defines the amount of cell fate determinants (e.g. Ascl1) and cell fate

Hypothesis of unequal inheritance of factors
* The distribution of cell fate determinants over two dividing cells defines cell fate

Question 25

What did Calegari and Huttner (2003) culture to investigate the effect of G1 lengthening in NSCs?

Answer 25

The authors used whole mouse embryo culture (WEC) of E9.5 mouse embryos.

Question 26

Why E9.5? Calegari and Huttner (2003)

Answer 26

Around E9.5 radial glia start to divide asymmetrically producing RG and neurons

Question 27

What mouse line was used and why?

Answer 27

A Tis21GFP transgenic mouse line is used (lineage tracing). Tis21 is a pro-neurogenic factor, specifically expressed in RG generating a neuron.

Question 28

What did they use to lengthen G1?

Answer 28

Olomoucine, a specific, cyclin dependent kinase (CDK1/2) inhibitor, lengthens G1 in
proliferating cells

Question 29

What did the authors find?

Answer 29

E9.5 Tis21GFP enbryos were grown 24 hours with Olomoucine:
* Upregulation of Tis21GFP in RG
* Marked increase of premature neurogenesis resulting in more neurons marked by MAP2

Question 30

What did the authors conclude?

Answer 30

Longer G1 results in RG differentiation

Question 31

Why would a longer G1 phase result in NSC differentiation?

Answer 31

During G1 cellular contents are duplicated. G1 is lengthened by activation of G1-specific CDK-inhibitors. By G1 lengthening more time is available for a cell fate determinant to be produced and transported to
the site of action. One of those cell fate determinants could be Ascl1, acting as a pro-neurogenic factor

Question 32

What does Marko question about this Calegari & Huttner (2003) study?

Answer 32

Calegari & Huttner (2003), Marko questions whether these red spots are cells, and they didn’t do checks whether these are neural stem cells. After this paper, nothing appears in the literature, leading to questions over how increases neurogenesis. How does a lengthening of G1 (through olomoucine) cause neurogenesis? Should question these figures as to what they are really showing. Could these be astrocytes?

Question 33

What method is often utilised to study how cortical diversity is achieved?

Answer 33

Lineage tracing

Question 34

Name an important marker for
a. Progenitors
b. Radioglia
c. Neurons
d. Neuroblasts (intermediate stage between iPCs and neurons)

Answer 34

a. Progenitors: Tbr2
b. Radioglia: Sox2
c. Neurons: MAP2/ NeuroD
d. Neuroblasts: EGFR

Question 35

Describe a study by Marten which used lineage tracing to study the development of the midbrain

Answer 35

Pitx3-Cre: Lox-STOP-Lox-YFP mouse to study Pitx3 expression

Pitx3Cre mouse: Cre is expressed in a Pitx3-dependent manner

LoxP-STOP-LoxP-YFP mouse: YFP is not expressed

By crossing the two models a mouse is created in which YFP is expressed in every cell which (on one timepoint or another) has been expressing Pitx3
Cells which expressed Pitx3, but have stopped Pitx3 expression
are still expressing YFP!

Daughter cells originating from a Pitx3 expressing cell inherit
YFP-expression because the genome has been changed in the mother cell and hence in all daughter cells!

Question 36

What finding of Marten’s was particularly unexpected?

Answer 36

Pitx3 expression in the eye was unexpected

Question 37

Describe the progression of studies investigating where all the different cortical cell types originate until the early 1900s

Answer 37

• Starting in the late 19th century: histological studies
• First analyses of brain tissue using the microscope
• Identification of different cell types based on morphological
  marks, ventricular zone is the most important germlayer
• Nissl-staining (cresyl violet) marks ER in neurons
• His (Nissl stainings, 1889): mitotic figures in the VZ
• Golgi-staining (silverchromate) marks neuronal cell bodies
• Ramon Y Cajal (Golgi stainings, 1909): distinction between neuroblasts and radial glia

Question 38

What was important about this finding in 1909?

Answer 38

Even back as far as 1909 there were recordings of neuroglia, they looked like neurons and had spikes but they did not really know what they are.

Question 39

What important contributions did Rakic add to this field in the 1960s - 1980s?

Answer 39

• 3H-thymidine (tritium) incorporation
• IHC/electron microscopy

3H-thymidine was developed which was radioactive (now use immunostaining).

Question 40

What did Kriegstein add to the field?

Answer 40

Novel methods allowing for accurate lineage tracing: cell culture, transgenic/viral
methods

Question 41

What did these developments by Rakic and Kriegstein allow for?

Answer 41

Through this they could show how the inner structure of the cortex was generated. Through this they could show how the ventricular zone was generated however at this time RG were seen as supportive cells.

Question 42

What did these novel methods confirm about old data?

Answer 42

Novel methods confirm old data: VZ is the most important germinal layer.

Question 43

How did the new methods update old data?

Answer 43

Radial glia were thought to be the scaffold of the developing cortex, in the mid 1960s it was thought Radial glia are scaffold and long-sought stem cells. With the availability of large screens for transcription and epigenetics new questions arise:
* Are radial glia progenitors predestined to become a certain type of neuron?
* How diverse is the cellular identity of cells in the cortex?

Question 44

What did Rakic propose first?

Answer 44

Rakic is an important author at this time, and was famous in the field alongside Kriegstein. Rakic was the first to propose radial glia were stem cells. He proposed that there was not only structure from the ventral to apical site but also functionally defined columns.

Question 45

What was the radial unit hypothesis?

Answer 45

This hypothesis postulates that the radial edifice of the cerebral cortex forms by a migration of vertically oriented cohorts of neurons generated at the same site in the proliferative ventricular zone (VZ) of the cerebral vesicle

Question 46

What implications does the radial unit hypothesis have for cortical diversity?

Answer 46

Therefore the generation of cortical diversity is time and place-defined; Cortical neural diversity is seen:
- Horizontally: columnar organization
- Vertically: layer formation

This diversity is linked to functionality

Question 47

How do inhibitory neurons arise in the cortex?

Answer 47

GABA-ergic, inhibitory interneurons are formed in the LGE (lateral geminal eminence) and MGE (medial geminal eminence) and migrate tangentially to the cortical plate before organising radially

Question 48

How do excitatory neurons arise in the cortex?

Answer 48

Glutametergic, excitatory projection neurons are born in the ventricular zone of the dorsal telencephalon, and migrate to their final position radially

Question 49

Give 6 functions of RG in the cortex

Answer 49

• scaffold for radial migration of pyramidal neurons
• pool of progenitor cells for self-renewal
• direct precursors of neurons
• direct precursors of IP (intermediate progenitors)
• direct precursors of astrocytes
• direct precursors of oligodendrocytes

Question 50

Therefore what relevance do RGs have for the development of the cortex?

Answer 50

Radial glia are of eminent importance for cortical development. Radial glia maintenance/differentiation is crucial in the evolution of the cortex

Question 51

What becomes of the neural plate and the intermediate zone following cortical development? What does it mean to say that these layers are time dependent?

Answer 51

The cortical plate will be the ‘brain layer’, the intermediate zone will become the white matter. The different layers are born at different time points.

Question 52

What cortical layers are present at E10.5?

Answer 52

Just the ventricular zone

Question 53

What cortical layers are present at E11.5?

Answer 53

The ventricular zone and the preplate (PP) above it

Question 54

What cortical layers are present at E12.5?

Answer 54

Preplate is split in marginal zone (MZ) and subplate (SP), forming the cortical plate (CP) between them

The ventricular zone adds the sub ventricular zone (SVZ) above it and the intermediate zone (IZ) between the SVZ and the SP

Question 55

What cortical layers are present at E14.5?

Answer 55

Layer VI appears between the SP and CP. The CP and IZ lengthen and the SVZ and VZ become smaller.

Question 56

What cortical layers are present at E16.5?

Answer 56

Layer V appears between the CP and VI. VI and IZ lengthen further and the SVZ and VZ shrink further

Question 57

What cortical layers are present in the adult?

Answer 57

Layer I, II/III, Iv, V, VI, SP and white matter

Question 58

Describe the birthdate of projection neuron subtypes in the VZ/SVZ from E11.5-E15.5 as shown in the diagram

Answer 58

Each shows a probability distribution peaking over the given time points:
E11.5: Subplate

E12.5: Corticothalamic/ layers VI callosal

E13.5: Subcerebral/ Layer V callosal

E14.5: Layer IV pyramidal

E15.5: Upper layer Callosal

Question 59

What broad description could be given to this pattern of development?

Answer 59

The cortex is build in an inside-out manner

Question 60

Which layers contain Cajal-Retzius cells?

Answer 60

PP and MZ (most apical layers) contain Cajal-Retzius cells

Question 61

Where does mitosis occur?

Answer 61

Only in the VZ and SVZ

Question 62

Where contains post-mitotic neurons?

Answer 62

IZ => CP

Question 63

When are the majority of pyramidal neurons born?

Answer 63

The majority of pyramidal neurons are born between E12.5 and E15.5

Question 64

When does synaptogenesis and formation of networks peak?

Answer 64

Synaptogenesis and formation of networks peaks around birth

Question 65

What are three important signalling pathways in this process and where are they most important?

Answer 65

• Reelin: PP
• Notch: VZ-SVZ (retain radioglia)
• IGF/Insulin: CSF (encourages proliferation)

Question 66

What does it mean to say that the generation of cortical diversity is time defined and what was used to study it?

Answer 66

Thymidine analogs (3HThymidine, BrdU, EdU) were used to study it as they stain for the S phase and you can see what neurons were proliferating when it was injected. Incorporation of radioactive Thymidine administered to a pregnant rhesus monkey, as
measured in the cortex of a young adult sibling.

Incorporation occurs mainly during the first half of pregnancy. The youngest neurons are positioned in the upper layers of the cortex. Early injection resulted in staining at lower layers of the cortex while later injections stained at higher layers of the cortex.

Question 67

Name the order of the first three neural cells to arise

Answer 67

Cajul cells are the first to rise, later the neuroepithelial cells and then later neurons.

Question 68

How is cortical layer formation regulated by reelin? What behavioural effects are seen in knockouts?

Answer 68

The preplate is usually split and neurons are generated between, this is not seen in reelin KO mice; instead of an inside out organisation you get an outside in organisation. This all has to do with the incorrect splitting of the preplate. Instead of the cortical layer forming between the PP and SP, it forms under them and cells of subsequent layers form in an unorganised manner under that. The cortical layers are therefore inverted so that L5/6 remains at the top and L2-4 form below it. The main hypothesis is that Reelin acts as a STOP/DETACH signal for migrating neurons at a “fixed” Reelin concentration to form these cortical layers.

Question 69

What is seen in born reelin knockout mice? Are the viable?

Answer 69

Reelin mouse was generated by accident when producing mouse lines, movement was impeded in an almost parkinsonian manner. These mice are still viable however and can breed, demonstrating the plasticity of the brain.

Question 70

Describe the effects of defects in four other genes which result in preplate disorders

Answer 70

Sox5: PP fails to segregate (partially); similiar to Reeler but the cortical layers form in the correct order under the MZ and SP (although more disorganised)

Tbr1: PP is partially split, SP is mispositioned to the middle of the CP (e.g between L5-6 and L2-4) and the layers are less segregated.

Satb2: migration of late-born neurons is delayed so that the SP is between the layers but they are organised. The defect is corrected postnatally

Pou3f2/3: PP is split but migration of L2-L5 projection neurons is stalled below SP so there is a thin layer of cells between the MZ and SP and the rest are below.

Question 71

What important process do these disorders highlight in brain development?

Answer 71

Interkinetic nuclear movement (INM) in NE and RG is crucial for normal brain development

Question 72

Describe the cell cycle of RG in relation to interkinetic nuclear movement

Answer 72

During G1 RGs migrate radially from the VZ towards the Pial surface. They move up, differentiate, change orientation, wait a few hours on top of the ventricular zone and wait to sense signals on the side of the cortex which can be dependent on the location of adjacent progenitor cells. Here they undergo the S stage. They then migrate back to the VZ while undergoing G2. At the VZ they undergo assymetric mitosis where a neuroblast splits and migrates apically to the pial surface and a progenitor remains at the neural tube lumen. This then repeats.

Question 73

Describe differences in transcriptional markers in RGs and neural progenitors (NPs) in this process

Answer 73

RGs: Sox2+/Tbr2-
NPs: Sox2-/Tbr2+/ Tuij+

Question 74

What are two differences in the division of RGs and IPCs?

Answer 74

RG divide asymmetrically in the VZ

IPC divide symmetrically in the SVZ

Question 75

How does this movement change in the last divisions of radioglia producing radioglia?

Answer 75

Radial glia final divisions produce translocating RG. They detach from the ventricle and begin to migrate apically along their processes, initially generating possibly nIPCs. These tRGs- translocating radial glia at the end of their lifespan; differentiate into astrocytes (earlier oligodendrocytes). This last division is not exactly known, there may be an intermediate cell involved or they may immediately transform into astrocytes

Question 76

Therefore describe the transitional process of NEs

Answer 76

• originate from the neural plate
• Divide symmetrical (self-renewal) several times forming NE
• Transform into radial glia

Question 77

Describe the transitional process of ventricular RGs

Answer 77

• originate from NE
• Divide asymmetrical several times forming RG+Neuron or RG+nIPC or RG+oIPC
• Transform into astrocytes or ependymal cells or adult neural stem cells (B-cells)

Question 78

Describe the transitional process of nIPCs

Answer 78

• Originate from RG
• Divide symmetrically forming neurons or neuroblasts (later B-cells)

Question 79

What disease is associated with improper interkinetic nuclear movement? The loss of which gene causes this?

Answer 79

Lissencephaly:
* Blocking of INM interferes with cell-cycle progression
* Loss of LIS1 (microtubule-associated protein) reduces cell proliferation

Lissencephaly occurs if this migration does not occur, there is an issue with the cell cycle and a smooth brain phenotype occurs.

Question 80

Describe two structures essential for mitosis

Answer 80

• The centrosome is an organelle serving as the main microtubule organising centre (MTOC). It is an important regulator of cell cycle and migration
• Microtubules are highly dynamic tubulin-based cytoskeleton components

Question 81

How are these two mitotic components involved in neural migration?

Answer 81

Motorproteins like kinesin and dynein are able to bind microtubules, regulating cellular processes like active transport, cell cycle and neuronal migration. The motor proteins pull the microtubules apart. The centrosome is positioned
ahead of the nucleus, with microtubules forming a perinuclear cage-like structure converging into the centrosome and projecting into the leading
process from the centrosome. microtubule structures couple (1) the leading process to the centrosome and (2) the centrosome to the nucleus to
translocate the nucleus. The centrosome is the part which is pulled up and the whole cell follows.

Question 81

How is Lis1 involved in this process?

Answer 81

LIS1 interacts with the cytoplasmic motor-complex dynein pathway. LIS1, Dynactin, Dynein, Doublecortin complex regulates nuclear movement:

Doublecortin is distributed to the perinuclear microtubule structure, and the Lis1–dynein (+ dynaction + doublecortin) complexes move in an microtubule minus end direction (minus at centrosome), attached to the nuclear membrane, to displace the nucleus toward the centrosome

If you lose Lis1, this migration of the nucleus and therefore migration of the cell does not occur.

Question 82

Describe five different cortical neuron subtypes

Answer 82

Corticofugal types:
Corticicothalamic: Project to thalamus
Subcerebral: Project to pons and spinal cord

Corticocortico types:
Columnar: Project to different layers of a column
Ipsilateral: Project to different columns and layers
Callosal: Project to the ipsilateral hemisphere

Question 83

Describe a study where they examined where this cellular diversity could arise from (Ayoub et al., 2011)

Answer 83

They isolated the ventricular zone, subventricular zone and cortical plate and did RNA-seq. They found that the dynamics of the transcriptional programs were well separated between the layers.

Question 84

What did this study using RNA Seq on different layers achieve and what was missing?

Answer 84

Creates markers of transcription factors for different layers but does not give different neuronal subtypes.

Question 85

Describe a study in 2015 which attempted to improve on this study

Answer 85

They did more or less the same but tried to be more specific in cell types using specific antibodies for three types of cells: callosal, subcerebral and corticothalamic. and pulled the cells out using the antibodies. They extracted the cells at different time points and sorted using FACS sorting based on the different colours onto different pots and carried out RNA Seq. These are quite specific to certain regions, the end goal is then to get transcription factors specific to each subtype in the different layers.

Question 86

What did they attempt to do with this RNA seq data (2015)

Answer 86

Generate a transcriptional map of the (developing) cortex. They identified trascriptional data about the different cell types and assorted them into a map in the cortex. This tells you about location, whether its up there and down there; which are important at different timepoints.

Question 87

What information does this give you about function? (2015)

Answer 87

Does not tell you function. If you see a group out genes going up at a different time point, you can look at what this could mean with a DAVID analysis. i.e what process might be involved such as cell cycle. If there is a certain gene you are interested in you can make a knockout to look at function.

Question 88

Name an alterative to knockout, What are the benefits (2) and drawbacks (2) in comparison?

Answer 88

Electroporation can be easier and cheaper than knockout and can choose a certain timepoint however you always knock it down and never completely knock it out.

Time can also be a disadvantage as you don’t know exactly how old it is or when the egg was fertilised. Therefore not all embryos are in the exact same stage of development when you carry out the electroporation.

Question 89

Describe the process of electroporation

Answer 89

You anaesthetise the mother, take out the uterus, you have to inject the ventricle in the little embryo using a light under the embryo. You need to inject a very small volume. You need to be trained, you can inject into the brain otherwise. You then need to use a small electrical charge to push the SiRNA or plasmid into the first or second layer into the brain. Cells along this pathway may then be affected by the SiRNA, to control for this you can use the scrambled RNA which should not interact with anything. You can target one layer but always in the same hemisphere, therefore you can utilise a control in the same hemisphere. You need to quickly electroporate or else it can leak into the opposite ventricle.

Question 90

Where are FOXO transcription factors expressed in the developing and adult brain?

Answer 90

FOXO6:
Corpus collosum
Hippocampus (CA1,3, DG)
Nucleus accumbens shell
Sulcus cinguli

FOXO1:
Dentate Gyrus (Intense expression)
CA3
Caudate putamen
Nucleus accumbens
Amygdalo hypothalmic fasculus
Posterior commissure

Question 91

What did they find following FOXO6 ablation?

Answer 91

Knew that it was expressed in the adult and in the embryo, didn’t know what it was doing i.e proliferation. Ordered a knockout and noticed a layering defect. they then electroporated GFP at E14.5; normally these radioglia become neurons, and saw clear difference:

In WT seen expression in VZ, SVZ and cortical plate. Seen more in VZ and less in SVZ and cortical plate in FOXO6 knockout although the order of the different layers was still maintained. Suggested that neurons were not migrating to higher areas; there were more in the deeper layers and less in the upper layers.

Question 92

What conclusions did they draw from this GFP FOXO6-/- study?

Answer 92

FoxO6 promotes migration of neurons born at E14.5

Question 93

What similar study did they follow up the FOXO6 -/- and why?

Answer 93

In utero electroporation of siRNA in E14.5 WT cortices: Not knocking out but similar with electroporation. Compensation factors could have rescued layering defects not observed earlier (layers staying the same order), however this shows that knocking it there at that time point has same effects and thus is unlikely to be a result of knockdown effects.

Question 94

How did they follow up this GFP FOXO6-/- study to investigate the mechanism behind these migration errors? Describe the methods

Answer 94

Took out the embryo, dissociated the brain and FACS sorted the GFP cells. Used two SiRNAs and one scrambled as a control. You can then sequence them both and compare them, only taking the overlap when comparing both to the scrambled RNA. In the DAVID analysis in C you can see that the same processes are regulated by the SiRNAs. Gives you an indication that you are targetting the same processes.

Question 95

Why use two SiRNAs for FOXO6?

Answer 95

Used two SiRNAs for FoxO6 as one Si may be more efficient than the other. This is both a control for the quality of the SiRNAs and the age of the embryos aka the timepoints. If you only take the overlap then you are sure these genes are really moderated by FoxO6.

Question 96

What did they find from this SiRNA transcriptomics study?

Answer 96

FoxO6 regulates Plxna4 in migrating neurons at E16.5 (PI3K is also regulated by FOXO6)

Question 97

What technique did they perform to further explore Plxna4 itself? What did they find

Answer 97

Did an ISH and found that it was expressed in the developing brain (Cortex + hippocampus)

Question 98

Describe how they further explored the relationship between FOXO6 and Plxna4 and what they found

Answer 98

They did a simultaneous in utero electroporation of siRNA and Plxna4 plasmid in E14.5 WT cortices and found that Plxna4 restores FoxO6 siRNA induced hampered migration. If you electroporate Plxna4 you see a rescue of the migration. Therefore Plxna4 is regulated by Fox06 and subsequently causes migration. Migrating neurons need Plxna4 which is expressed by FoxO6.

Question 99

How does FOXO6 regulate Plxna4?

Answer 99

FoxO6 binds and regulates the Plxna4 promoter

Question 100

What signal cascade is Plxna4 part of?

Answer 100

The Plexin/Semaphorin signalling cascade: axon guidance factors involved in the development of the neuronal system. Upon binding of semaphorin to the extracellular region, plexin is activated and transduces signal to the inside of the cell through its cytoplasmic region.

Question 101

What did they observe in FOXO6 deficient neurons at P7? (3)

Answer 101

Following in utero electroporation of GFP plasmid in E14.5 WT and FoxO6 -/- cortices, they noticed FoxO6 deficient P7 neurons are stranded in L5/6.

Additionally, FoxO6 deficient P7 neurons change identity

FoxO6-/- neurons display altered morphology

Question 102

In what way did FoxO6 deficient P7 neurons change identity? How did they check this?

Answer 102

They used: to stain for
Ctip2: L5/6 marker
Satb2: callosal neurons
Cux1: L2/3 marker

They found that FoxO6 -/- P7 brains show a decreased number of Cux1 expressing pyramidal neurons and Satb2 expressing callosal neurons

Question 103

Describe the changes in morphology

Answer 103

The GFP+ neurons in WT were angled more radially with their processes pointing apically. The GFP+ neurons in FOXO6 -/- were angled more tangentially with their processes pointing bilaterally.

Question 104

What stuctural changes related to these differences in neural identity were noted? What did they take from this?

Answer 104

FoxO6 -/- or deficient cortices exhibit smaller Corpus Callosum: FoxO6 is involved in Corpus Callosum development

Question 105

How is this structural difference in FOXO6 ablation relevant to the transcriptional analysis?

Answer 105

FoxO6 also regulates both Nfia and Nfib in E16.5 migrating neurons. The ablation of either of these leads to Corpus Callosum agenesis, to a larger extent in Nfia.

Question 106

Does Plxna4 restore this defect? How can it be restored?

Answer 106

No, Plxna4 restores FoxO6 siRNA induced hampered migration but does not restore Corpus Callosum defect. FoxO6 siRNA induced CC defect is only rescued by simultaneous expression of Nfia and Nfib

Question 107

Therefore summarise the roles of FOXO6 in neurodevelopment

Answer 107

• FoxO6 regulates Plxna4 in order to ensure correct cortical migration of E14.5 born neurons
• Hampered embryonal migration in FoxO6 silenced cortices leads to a changed microcircuit assembly
• FoxO6 is necessary for a normal Corpus Callosum formation via regulation of both Nfia and Nfib
• Plxna4 rescues neuronal migration, but does not restore CC defect in FoxO6 siRNA treated cortices
• FoxO6 promotes adult neural stem cell activation in a FoxO3 dependent manner