Notch Signalling in Cortical Development

Question 1

How variable is the notch signalling pathway across species?

Answer 1

Notch signaling pathway is highly conserved in evolution

Question 2

Describe the Delta-Notch signalling pathway

Answer 2

Interaction between the Delta cell surface ligands and their Notch receptors on neural progenitor cells in close proximity to one another regulates transcription factors necessary for the generation of differentiated neurons.

Delta binding cleaves a protein fragment of the receptor (the Notch intracellular domain, or NICD). When the NICD is transported into the nucleus, it binds to a transcriptional complex including the recombining binding protein J (RBP-J), inhibiting RBP-J repression and resulting in the transcription of (among others) the Hes family of transcription factors, including the bHLH neurogenic factors responsible for neuronal differentiation.

Question 3

Describe the relationship between Delta ligands and differentiation

Answer 3

Delta, Notch, and bHLH proteins are expressed at similar levels in a cluster of progenitor cells and neuroblasts. A stochastic increase in Delta ligands on a particular cell leads to downregulation of Delta in neighboring cells, while in the Delta-upregulated cell, bHLH gene expression is also upregulated, and the cell becomes primed for neuronal differentiation.

Question 4

How many Notch receptors do Mammals have?

Answer 4

Mammals possess 4 Notch receptors (Notch1,2,3,4)

Question 5

What kind of receptors are notch receptors? What happens when notch ligands bind to them?

Answer 5

Notch receptor is a hetero-oligomer transmembrane protein. Notch ligands bind to the extracellular domain and cause proteolytic cleavage and release of the intracellular domain

Question 6

What are most Notch ligands? What does this mean for function?

Answer 6

Most Notch ligands are transmembrane proteins, making Notch-signaling a typical cell-cell interaction pathway

Question 7

Is Notch more in development or in mature brains?

Answer 7

Notch function is highly versatile, ranging from neural development to mammary gland regulation and cancer development

Question 8

What is Notch signalling most known function

Answer 8

Cell fate decisions

Question 9

In what part of corticogenesis is notch signalling particularly important for?

Answer 9

Notch signaling is involved in corticogenesis especially during neurogenesis

Question 10

Describe the normal pathway for notch in stem cell maintenance by the diagram used in the slides

Answer 10

It is involved in corticoexpansion: Mash1 and NGN2 activate Delta1 (Dll1) which with Mindbomb1 create the DII1 transmembrane protein.

When this interacts with the Notch receptor the NICD protein is detached and travels to the nucleus. Numb, however can inhibit it at this time.

NCID then forms a complex with Rbpj and MAML (mastermind-like) to activate the Hes1/Hes5 gene. These proteins inhibit Mash1 and Ng2.

Question 11

What is the relevance of this inhibitory effect?

Answer 11

It carries out lateral inhibition. Lateral inhibition (in neural development) is a cell-cell interaction whereby
a cell adopting a certain fate, inhibits its immediately neighbours from doing likewise

Expression of Delta is important for differentiating cells, cells which are differentiating express Delta. There is lateral inhibition, it regulates its own expression. The bHLH genes, Mash1/ngn2 are downregulated by Hes1 and Hes5 and therefore do not activate delta to incur notch signalling in surrounding cells.

Question 12

What pattern does this signalling dynamic incur in RGCs

Answer 12

Expression of Notch effectors (Hes1/Hes5) is highly dynamic in neuronal progenitors and Notch activation levels in RG exhibit an oscillatory pattern:

Notch activates the Hes1 gene, resulting in transcription of Hes1 RNA and translation of the Hes1 protein. This protein then represses further transcription of the gene and is degraded through the upiquitin-proteosome pathway.

Question 13

What does the Smidt lab also think shows this oscillatory pattern?

Answer 13

They also think that Foxo6 may be oscillating, however this is very hard to show in the lab.

Question 14

What downstream effects does this oscillitory pattern have?

Answer 14

There is dynamic expression of Notch effectors in neural progenitors. Ngn2 and DII1 expression oscillates in a pattern directly opposing a pattern of Hes1.

Question 15

How does this pattern differ in a stem cell to a neuron?

Answer 15

In neural stem cells/ progenitors this expression pattern is oscillating. The decision to remain a stem cell is this balance and when this balance is disturbed then differentiation occurs. Differentiation occurs when Ngn2 and DII1 remain high, when notch signalling occurs, and Hes1 remains low.

Question 16

Relate Notch and delta levels to the aforementioned migratory patterns

Answer 16

Notch and Delta levels seem interconnected with cell cycle and interkinetic nucleus migration.

Notch is high at the apical site and delta is high at the basal site.

Notch is therefore high at G1, when the NPC is closer to the apical site.

Delta is then high in the S phase, when the NPC is at the basal site.

Notch becomes higher as the cell migrates to the apical site again while going through G2 and is the highest as it undergoes mitosis.

Question 17

Describe a way in which notch aids differentiation

Answer 17

It is involved in asymmetrically localised cell fate determination. mPar3 shows a dynamic distribution in RG during cell cycle, resulting in asymmetric cell division during the neurogenic phase. mPar3 and Notch signaling are interconnected in a regulatory feedback-loop

Question 18

In the paper which described this mPar3 Notch feedback loop, what was the main question?

Answer 18

how is asymmetric cell division regulated in radial glia?

Question 19

What were the methods of the mPar3 Notch paper?

Answer 19

Study was performed in WT and Par3 deficient mice and it utilised IHC, neocortical slice culturing, Cre-mediated ectopic gene-expression.

They did a clonal cell pair assay in which embryos at E.13 were removed from the womb and coronal slices were obtained. At E.14 cortical tissue was plated at clonal density and cultured for 24hrs before fixation and IHC.

Similarly for neocortical slice culturing they cut a section of the dorsal cortex and underwent organotypic slice culture before doing time-lapse imaging for 24 hours. They then fixated the tissue and underwent IHC.

Question 20

What is confocal microscopy?

Answer 20

Confocal microscopy focuses the light at what you are looking at and filters the light being reflected. This allows you to look at different layers of cells rather than just looking at the tissue.

Question 21

What did the mPar3 Notch paper find?

Answer 21

They found that the mpar3, as well as ZO-1 (junctional protein) expression was enriched at the luminal surface of the VZ. It found they largely colocalised with each other at the lateral membrane domain in the ventricular endfeet. Through further staining they found that mPar3 and ZO-1 were expressed together at the apical ends of the end feet (towards ventrical) which B cantenin and N-cadherin (junctional proteins) were expressed independently higher up on the end feet.

Question 22

What is meant by these end feet?

Answer 22

Cell body of radioglia is attached to the ventrical via an endfoot; the cell body is not attached to the ventricle. The nucleus is also in the endfoot; it is not always in a rounded structure

Question 23

What does it mean to say that RGs are polarised?

Answer 23

Cortical progenitors (RGs) are highly polarized, facing the lumen of the ventricular zone, and reaching to the pial surface via radial processes

Question 24

How is apical basal polarity maintained?

Answer 24

Apical-basal polarity is maintained by adherens junctions (cell- cell adhesion)

Question 25

Aside from maintaining polarity, what further functions do adherens junctions have?

Answer 25

• Adherens junctions contain cadherins, which connect to catenins, together regulating actin filaments dynamics
• Actin dynamics asymmetrically distribute proteins in progenitor cells
• Asymmetric distribution of proteins modulates asymmetric cell division

Question 26

What does the deletion of alpha-cantenin result in?

Answer 26

Deletion of alpha-catenin results in acclerated proliferation and hyperplasia (increased cell production in a normal tissue or organ)

Question 27

What effect does alpha-cantenin have on beta-cantenin?

Answer 27

alpha-catenin positively modulates beta-catenin

Question 28

How are the components of adherens junctions otherwise modulated? What relevance does this have for RGs?

Answer 28

The components of adherens junctions are tightly linked to a variety of signal transduction pathways (Wnt/Shh). The units connecting these junctions between radioglia is therefore regulated by Wnt signalling. There is unequal distribution because these proteins move between cells using these junctions

Question 29

How do these adherens junctions regulate these protein distributions?

Answer 29

Adherens junctions regulate the Intracellular distribution of so-called apical protein complexes by forming a “docking station.”

The unequal distribution of proteins modulates the orientation of the mitotic spindle thereby determining if a progenitor divides symmetrically or asymmetrically

Question 30

Therefore name four things apical protein complexes in apical neural progenitors interact with

Answer 30

• adherens junctions
• Actin filaments
• Notch effectors Numb and Numbl
• Components of Wnt and Shh signaling

Question 31

Where are apical compleses present?

Answer 31

only present in VZ-based progenitors

Question 32

How varied are apical complexes across species?

Answer 32

Apical complexes are evolutionary conserved and were first studied in C.elegans and Drosophila

Question 33

Name the most studied apical complex in mammals

Answer 33

Par3/Par6/PKC complex

Question 34

In what way do apical complexes interact with Numb?

Answer 34

Apical complexes regulate the distribution of
* Numb in invertebrates (Drosophila and C. Elegans)
* Numb and Numbl (Numb-like) in vertebrates

Question 35

What is the function of Numb? What happens in deletion?

Answer 35

Numb and Numbl are required for continued cell division, deletion leads to premature exit from the cell cycle (thin neocortex and small hippocampus)

Question 36

Name two other things Numb interacts with

Answer 36

Numb and Numbl interact with adherens junctions, deletion leads to mistargeted cadherins and disruption of junctions

Numb inhibits Notch signaling by blocking nuclear translocation of NICD

Question 37

Describe the interaction of apical protein complexes and Numb, what consequenc does this have

Answer 37

Numb is phosphorylated in a Par3/Par6/PKC dependent manner, phosporylated Numb then associates with the lateral membrane domain of ventricular endfeet.

Associated (bound) Numb is not able to negatively regulate Notch-activity. In general, daughter cells with large amounts of Par3 and inactive Numb will maintain progenitor cell identity

Question 39

Now, relate this back to the study analysing Notch effect on differentiation

Answer 39

Dynamic distribution of mPar3 in radial glial cells leads to differential inheritance of mPar3 by the two daughter cells as the cell cycle progresses. The daughter cell that inherits a greater amount of mPar3 develops high Notch signaling activity and remains a radial glial cell, whereas the daughter cell that inherits less mPar3 harbors low Notch signaling activity and adopts an IPC or a neuronal fate.

Question 40

describe the molecular contents of a VZ RG assymetrically dividing

Answer 40

The side remaining an RG likely contains Par3/6/PKC and Notch, while the side differentiating likely contains higher delta like 1 (Dll1),