Neural stem cells, early brain development, cell cycle

Question 1

What two hypotheses were there when they wanted to explore FOXO6’s role in quiescence and activation of stem cells?

Answer 1

h1: FoxO6 ablation leads to decreased activation of quiescent stem cells

h2: FoxO6 ablation leads to increased return to quiescence of active stem cells

Question 2

Why did they decide to focus on FoxO6?

Answer 2

A pioneer vector is an important TF which affects the chromatin. They think Fox06 may be this pioneer vector not only regulating the other FOXOs but also other TFs. FOXO6 could modulate 03 and therefore modulate quiescence and activation.

Question 3

What can be used to analyse the structure of chromatin?

Answer 3

ATAC-seq can be used to assess the structure of the chromatin.

Question 4

What is often done instead of chip-seq now? Why?

Answer 4

Cut and run is often now used instead of ChIP, you IP the protein in combination with enzymes which are bound to your DNA protein. The enzymes cut a couple of nucleotides from your binding sites. ChIP is very difficult if you don’t have an antibody of an appropriate grade.

Question 5

Describe how they explored the role of FOXO6 in this transition

Answer 5

They knocked out foxo6 in a P60 mouse and retrieved stem cells from the dentate gyrus of both that and a control animal. They then cultured the cells into neurospheres.

They used EdU staining which is a marker for DNA replication in the S phase of the cell cycle to see when proliferation is occurring. This was added 1 hour before cells were fixed at the following time points, note that other labs did not replicate findings by other labs. They incubated both groups with just FGF2 for 24hrs, T1. After 72 hours they incubated them with BMP4 (promotes the cell cycle and differentiation at low doses, decreases at high doses) 72hrs after T1, T2. Then the following three timepoints (24hrs, 48hrs, 72hrs) they incubated them with FGF2’, T1 T2 T3.

Question 6

What effect did BMP4 have?

Answer 6

BMP4 induced decrease of proliferation. In the first time point the number of EdU positive NSCs would be around 20%, plummet when BMP4 was added and then rise for the following two time points before stabilising at the last time point.

Question 7

What did they observe in their experiment?

Answer 7

In control condition they noted a similar pattern with a drop in EdU+ cells after BMP4 and a rise and then slight drop in the last timepoint.

In the FoxO6-/- condition they noted a much more dramatic decrease following BMP4 incubation and a slow rise following this in the subsequent timepoints.

Question 8

What conclusion did they draw from this?

Answer 8

P60 AHNSCs WT vs. KO: KO does not reach 100% reactivation after 72h; This suggests that cells are more easily pushed into quiescence without Fox06 as EdU positive cells drop at a higher rate following BMP4 administration.

Question 9

More time points could be taken here to draw more conclusions over longer time points. What would be a negative consequence of this?

Answer 9

The density of the cells however could lead to stress, cell death or other effects. There is contact inhibition, they contact each other and stop the cell cycle and therefore stop dividing and eventually die. (The issue with getting oxygen and nutrients is more with organoids and neurospheres.) They could be transferred to another disc but this would change the environment.

Question 10

Describe the process of neurulation

Answer 10

1. Neuroectodermal tissues differentiate from the ectoderm and thicken into the neural plate. The neural plate border separates the ectoderm from the neural plate.
2. The neural plate bends dorsally, with the two ends eventually joining at the neural plate borders, which are now referred to as the neural crest.
3. The closure of the neural tube disconnects the neural crest from the epidermis. Neural crest cells differentiate to form most of the peripheral nervous system.
4. The notochord degenerates and only persists as the nucleus pulposus of the invertebral discs. Other mesoderm cells differentiate into the somites, the precursors of the axial skeleton and skeletal muscles.

Question 11

Describe the process of neuroepithelial cells (NE) to ‘B cells’ (adult NSCs of the SVZ) in the cortex

Answer 11

NEs develop into early radial glial cells which can first assymetrically divide (AD) directly into neurons, or indirectly through nIPCs.

Slightly later they can AD into oIPCs (oligodendrocyte intermediate progeitor cells) and then slightly later into astrocytes or aIPCs.

In between oligodendrocytes and astrocytes it is postulates that a subset may develop into SGZ radial astrocytes and remain in the hippocampus and from there they can AD into neurons from nIPCS and possibly oIPCs and astrocytes.

Late radial glial cells in the SVZ also develop into ependymal cells or B cells (Adult NSCs). B cells can generate nIPCs, oIPCs and possibly astrocytes or other B cells.

Question 12

Name an aspect in this develop that is more important for humans compared to a lot of other animals

Answer 12

nIPCs: neuronal intermediate progenitor cells, this intermediate step is the most important in human development.

Question 13

What role do the first neurons play in the formation of the cortex?

Answer 13

These first neurons are formed directly from radial glia, these neurons stay at the top of the cortex; they then send stop or migratory signals to newer neurons before later disappearing.

Question 14

How do microglia arise?

Answer 14

Microglia invade the brain and are formed from the marrow, technically they are not neural cells.

Question 15

What are the first neural stem cells to appear?

Answer 15

Neuroepithelial cells in the neural plate and tube: NEs, early neural precursor stem cell

Question 16

What is their make-up and behaviour?

Answer 16

Epithelial make-up, amplification by symmetric divisions

Question 17

Describe the junctions of NEs

Answer 17

Highly polarized, tight junctions

Question 18

What are the markers of NEs? (6)

Answer 18

Sox2
Sox10
Notch1
Hes1 and 3
E-cadherin
Occludin

Question 19

How do layers form from these first NEs?

Answer 19

• Neural tube first consists of one layer of highy polarized NEs
• More layers are created by asymmetric divisions
• Stratification increases as apical-basal polarity is decreased

Question 20

What is meant by inductive signalling in the neuroepithelial stem cell niche?

Answer 20

The influence from one cell group over a
neighbouring cell group during development or the controlled orchestration of intrinsic developmental
programs governed by extrinsic signals

Question 21

Name four inductive centers and their signals

Answer 21

Roofplate: BMP, RA, Wnt
Floorplate: Chordin, Shh, RA, Wnt
Somite: BMP
Notochord: Shh

Question 22

How many Wnt ligands are there and what pathway does it activate?

Answer 22

The 19 human Wnt ligands (encoded by 19 separate genes) can activate two distinct signal transduction cascades, the “canonical” and “non- canonical” pathways.

Question 23

Describe the canonical pathway

Answer 23

The noncanonical Wnt pathway regulates cell movements necessary for lengthening the neural plate and neural tube. Here Wnt ligands activate receptor proteins (Frizzled), leading to changes in intracellular Ca2+ levels; alternatively, the Wnt ligands can bind an orphan receptor tyrosine kinase, leading to activation of a Jun kinase (Jnk) signaling pathway that can phosphorylate several intracellular targets, leading to changes in cell shape and polarity

Question 24

Describe the canonical pathway

Answer 24

The canonical Wnt pathway influences cell proliferation, adhesion, and differentiation after the initial morphogenesis of the nervous system (gastrulation and neurulation) is complete. This pathway relies on the activation of the Frizzled receptor in the presence of a co-receptor (Lrp5/6) which leads to stabilization of b-catenin, a cellular messenger which is then translocated to the nucleus, where it influences gene expression via interactions with the TCF/ LEF transcription factor

Question 25

What do the slides say that each pathway is important for?

Answer 25

• Canonical pathway (gene transcription)
• Non-canonical pathway (cytoskeleton
  dynamics and internal calcium regulation)

Question 26

What kind of hormone is Wnt?

Answer 26

peptide hormone

Question 27

What do the slides say Wnt signaling is important for?

Answer 27

• Body axis patterning
• Stem cell maintenance
• Regulation of asymmetric division
• Neural migration
• Neural differentiation

Question 28

What kind of hormone in FGF and what ligands does it encompass?

Answer 28

Peptide hormone which has among the largest sets of inductive signals
- 22 diffent ligands, coded by 22 genes

Question 29

Describe the FGF signalling pathway

Answer 29

All of these ligands bind to the same receptor tyrosine kinases (FGFr) that initiate a phosphorylation-based signaling cascade via the RAS/MAP kinase pathway which can regulate genes via transcription.

Since there are different ligands for FGF which different functions, there is competition between ligands. Different ligands have functions via different effects following activation of the receptor.

Question 30

What is FGF mainly involved in?

Answer 30

Mainly involved in cell proliferation
and differentiation

Question 31

What is FGF8 mainly involved in

Answer 31

FGF8 is known for its role in early
forebrain development

Question 32

What does basic FGF promote in stem cells?

Answer 32

Basic FGF promotes stemness in stem cells

Question 33

What is RA? How is this relevant to its functions?

Answer 33

RA is a metabolite of vitamin A (member of the steroid/thyroid superfamily of hormones); RA signaling mediates vitamin A functions

Question 34

Describe the RA pathway

Answer 34

RA activates a unique class of transcription factors that are also receptors for RA and related ligands—the retinoid receptors. When activated by RA or related retinoids, the ligand-receptor complex modulates the expression of several target genes. The capacity of RA receptors, when bound by RA, to stimulate or repress gene expression depends on co-activators or co-repressors that form complexes with the receptors when bound to nuclear DNA (requires these nuclear receptors).

Question 35

Name three things which require RA signalling

Answer 35

• Required for growth and development
• Modulates Hox-gene expression
• Necessary for anterior/posterior axis

Question 36

What initiates inductive signalling and what effects does this have for a stem cell?

Answer 36

A combination of intrinsic programs
and extrinsic signals initiates
Inductive signaling. This in turn, leads
to the expression of transcription
factors, which determines cell identity

Question 37

What roles do BMPs play?

Answer 37

The bone morphogenetic proteins (BMPs), are particularly important for a variety of events in neural induction and differentiation. The various BMPs play roles in the initial specification of the neural plate as well as the subsequent differentiation of the dorsal part of the spinal cord and hindbrain and the cerebral cortex.

Question 38

Describe the signalling pathway of BMP

Answer 38

In humans and other mammals, six distinct genes encode six different BMP ligands. These ligands all activate a singular signaling pathway via the same receptor serine kinases, resulting in the phosphorylation and translocation to the nucleus of transcriptional regulators called SMADs. Following BMP-dependent phosphorylation, three different phospho-SMADs—1, 5, and 8—translocate into the nu- cleus, bind to specific enhancer/supressor DNA sequences called BMP response elements (BMPre) and thereby influence transcription of several target genes.

Question 39

In mesodermal cells, the BMPs elicit osteogenesis. When ectodermal cells are exposed to BMPs, they assume an epidermal fate, forming structures associated with the skin. How, then, do ectodermal cells become neuralised, given that BMPs are secreted by the somites and surrounding mesodermal tissue?

Answer 39

The mechanism evidently relies on the local activity of additional secreted inductive signaling molecules, including Noggin and Chordin—two members of a broad class of endogenous antagonists that modulate signaling via the TGF-b family (including the BMPs). These antagonistic molecules can bind directly to BMPs, preventing their binding to BMP receptor proteins. When BMPs are thus blocked from their “normal” receptors, neuroectoderm is “rescued” from becoming epidermis and continues along a path of neuralisation. This negative regulation has reinforced speculation that becoming a neuron is the “default” fate for embryonic ectodermal cells.

Question 40

So therefore are BMPs important for neuronal differentiation?

Answer 40

Yes, once neuronal precursor identity is firmly established, however, BMPs can act on neural precursors or differentiating neurons to further influence their identity and fate.

Question 41

Describe the effects of of BMP ligands in the neural tube as they are released from the roof plate

Answer 41

The neural progenitor cells are first divided into broad dorsoventral domains by the differential expression of Pax homeobox genes. BMPs activate the dorsal
Pax genes and set the expression domain boundary of Pax6 by repressing it in the dorsal spinal cord.

The spinal cord is further subdivided by the expression of basic helix–loop–helix (bHLH) family and homeodomain transcription factors. This process is also tightly regulated by BMP signalling. High levels of BMP signalling set up the most dorsally located Math1 domain and lower levels of signalling define the less dorsal cell populations, which express neurogenin 1 and 2 (NGN1/2) and the transcription factor Mash, respectively.

The regionalisation of the precursor cells leads to formation of distinct dorsal and ventral neuronal cell types, namely the dorsal interneurons (dI1–6), the ventral interneurons (V0–3) and the motor neurons (MN).

Question 42

What do the slides say about dorsal and ventral identity promotion?

Answer 42

Dorsal identity is promoted by BMPs and TGF-B

Ventral identity is promoted by Sonic Hedge Hog and Chordin. Depending on the relative concentration of chordin and SHH at the floorplate, identity is determined.

Chordin is an antagonist of Bmp signaling

Question 43

Describe the diagram depicting the transition from neuroepithelial cells to neuronal diversity in terms of signalling

Answer 43

Neural Induction: The ectoderm in the presence of BMPs becomes the epidermis. In the presence of Noggin/ Chordin it becomes the neuroectoderm.

Organiser centers: The neuroectoderm forms into the neural tube and TGF-Bs for the dorsal centers while SHH forms the ventral centers.

Neural patterning: There is a difference in sigalling from the dorsal and ventral domain.

Neurogenesis: Notch inhibit (Notch signals are required to maintain stem cells so for differentiation it must be inhibited), and bHLH gene products activate neuronal precursors which become neurons.

Oligogenesis: Proneural bHLPs inhibit, and Olig 1/2 and Nkx2.1 activate oligodedrocyte precursors which become oligodendrocytes.

Astrogliogenesis: Proneural bHLPs inhibit, and Notch/ Nrg activate astrocyte precursors which become astrocytes.

Question 44

Describe three morphological or transcriptional changes as neuroepithelial cells transform into radial glia

Answer 44

Bipolar morphology is conserved, but tight junctions are lost

Transcriptional identity changes from epithelial to glial

Astroglial markers like GFAP, GLAST and Blbp are expressed in RG

Question 45

What supports elongated radioglia?

Answer 45

GFAP and GLAST are cytoskeleton proteins providing strenth to support elongated RGs

Question 46

How do human RGs differ from mouse RGs?

Answer 46

human RGs differ from mouse RGs in
-location
-developmental potential
-molecular make-up

Question 47

What are the first neurons to form and what signals do they receive?

Answer 47

These first neurons are the cajal retzius cells, which receive reelin signals.

Question 48

How do interneurons arise?

Answer 48

Interneurons migrate tangentially from the ganglionic eminence of the basal telencephalon to the pallium to disperse throughout the cerebral cortex. The tangentially migrating interneurons travel perpendicular to the radial glial cells. The radially migrating interneurons travel parallel to the radial glial cells.

Question 49

How do human and mouse RGs in the differ in terms of location?

Answer 49

The oldest neurons that remain in the mouse cortex are in the bottom. In humans this is different, at the bottom there is also an outer ventricular zone full of stem cells which generates more stem cells and is quite larger. It generates the majority of the cortical stem cells.

Question 50

How is neuronal identity determined in cortical development?

Answer 50

In cortical development neuronal identity is determined by local inductive signaling (niches) and the specific expression of transcription factors. This inductive signalling and the expression of transcription factors is time/place dependent. The final identity of all cells in the cortex (neurons and glia) is defined by the timepoint and place of birth. Transcriptional dynamics (time and place specific expression of transcription factors) ultimately governs neuronal diversity in the brain

Question 51

Describe where different signals arise in the RG stem cell niche

Answer 51

RA and BMPs are secreted from the meninges

Surrounding IPCs and neurons secrete Mib1/Delta and Sip1/Ntf3

RGs secrete Notch and Fgf1R

Cerebrospinal fluid secretes Igfs, Fgfs, Wnts, TGF-B/BMPs, RA and Shh

Question 52

To what extent are neurons along cortical development in contact with basal and apical structures?

Answer 52

NEs and RGs are in direct contact with the CSF (basal) and pial surface (apical). Basal progenitors do not contact the CSF or pial surface. higher OSVZ radial contact the pial surface but do not contact CSF. Neurons form the cortex and do not contact either.

Question 53

What is the significance of these progenitor cells being in contact with the CSF?

Answer 53

Each RGC projects a small cilia into the ventrical where factors distributed by the CSF can have an effect. NE and RG cells receive signals from the CSF via cilia, the content of the CSF changes considerably over time; the contents of the CSF at 14.5 compared to 18.5 are completely different.

Question 54

What other function do the cilia have?

Answer 54

They create a flow in the ventricals, they simultaneously create a flow and sense the contents.

Question 55

What functions does the CSF have?

Answer 55

The CSF provides protection through buoyancy, structure through pressure, flows out waste and provides growth factors, insulin etc.

Question 56

What controls what goes in and out of the ventricals?

Answer 56

Ependymal cells line the ventricles and are similar to epithelial cells. The ependymal cells control what is going in and going out.

Question 57

Describe the CSF flow route in the embryo and adult

Answer 57

In the embryo the flow has to go through the telencephalic vesicle to the mesencephalic vesicle to the rhombencephalic vesicle.

In the mature CNS, CSF generated primarily by the choroid plexus tissues located in each ventricle in the brain fills the ventricles, subarachnoid space, and spinal canal. CSF flows from the lateral ventricles via the foramen of Monro/intraventricular foramen into the mesencephalic/third ventricle, and then via the aqueduct of Sylvius/cerebral aqueduct into the hindbrain/ fourth ventricle. The CSF then continues through the foramina of Magendie/median apertures and Luschka/lateral apertures into the spinal canal and subarachnoid space, and is finally resorbed into the venous system via arachnoid villi.

Question 58

How is CSF formed?

Answer 58

CSF is formed by the choroid plexus, which contains all the ependymal cells of an adult brain. The choroid plexuses are present in the two lateral, third and fourth ventricles. Each of the plexuses is comprised of fenestrated vessels, with a single layer of intimately opposed choroid epithelial cells, joined by tight junctions—forming the blood-cerebrospinal fluid barrier.

The capillary forms a U shape and is held together by connective tissue of pia mater. The capillary is lined with ependymal cells. Wastes and unnecessary solutes are absorbed from the vesicle into the blood vessels and CSF forms
in the ventricle as a filtrate through the capillary and ependymal cells containing glucose, oxygen, vitamins and ions.

Question 59

How can you analyse CSF function? (4)

Answer 59

• By using mouse models (cilia-mutants) (also KO flow)
• By injecting antibodies or growth factors into the CSF (but can create aggregates)
• By ex vivo cultures and adding relevant substrates (Like little brains)
• By analysis of CSF-content (Western, MassSpec): proteomics

Question 60

Describe mass spectrometry

Answer 60

• An analytical technique that produces spectra of the masses of molecules comprising a sample of material. In biology, the spectra are used to determine which peptides are in the analyzed sample
• A sample is first treated with trypsin (proteins are fragmented), then ionized and separated according to mass-charge ratio, and finally
  detected by a mechanism able to detect charged particles
• The measured spectra are compared to known spectra and molecules are identified (MASCOT)

Question 61

What is a difficulty when analysing CSF? (2)

Answer 61

You can only get around 1ul of CSF from an E14.5 embryo and it is very difficult to collect this without also cells.

Question 62

Describe the process of MS depicted in the diagram

Answer 62

Sample preparation and fractionation can be done with SDS PAGE or 2D gel electrophoresis. Proteins can the be digested with e.g trypsin. The peptides can then be separated with HPLC or ion exchage.

Once a sample s injected a heater can vaporise the sample and an electron beam ionises the sample. The particles are then accelerated into a magnetic field which separates them based on their mass/ charge ratio onto a detector.