Lecture 7 - Developmental Neurobiology - Neurogenesis

what is neurogenesis?

• how neurons are born from progenitor cells

what can cell fate be affected by?

• affected by orienting plane of cell division

do all progenitor cells become neurons?

• not all - some progenitors stay as progenitors next to the lumen in a region called the ventricular zone (VZ)

what are the 3 types of cells that progenitor cells can give rise to?

1. neurons

2. astrocytes

3. glia

what is a radial glial cell?

• a stem like cell whose nucleus/cell body is at the lumen, and has a long basal process

when the Shh pathway is activated what do the Gli activators do?

• Gli activators go into the nucleus and transcriptionally upregulate a gene

what lines the early neural tube?

• only 1 cell wide

• the neural tube is lined with neuroepithelium/neuroepithelial cells

• these are progenitor cells that are patterned by BMPs or Shh

why does the neural tube look as if it’s several cells wide?

• the progenitor cells are undergoing mitosis - the cells are at different stages of the mitotic cycle so it appears layered

• due to the movement of cells as they undergo mitosis - the nucleus is migrating in the cells

• this gives the appearance that the neural tube is several cells wide

what is interkinetic nuclear migration?

• the movement of the nucleus within neural progenitor cells in the pseudostratified neuroepithelium of the neural tube in coordination with the cell cycle

explain the cell cycle of the nuclei of the progenitor cells

1. in G1 and S phase - the nucleus is away from the lumen

2. during G2 - the nucleus moves back towards the lumen

3. at M phase and cytokinesis - the nucleus is close to the lumen - and the cell divides there

4. at cytokinesis - the lateral attachment is lost and then reformed

how can the early neuroepithelial cells divide?

• they can divide only symmetrically

• generating two identical daughter cells

what do early neuroepithelial cells become?

• they change shape and become radial glia like cells

what are the 2 ways that radial glia can divide?

1. symmetrically

2. asymmetrically

what is the symmetric division of radial glia?

• a radial-glia can divide to give more radial glia

• resulting in production of 2 identical radial glia

• self-renewal

what is asymmetric division of radial glia?

• 1st daughter - identical to the mother (radial glia) stays at lumen

• 2nd daughter - acquires a neuronal identity = differentiates into a neuron

  → this daughter uses the scaffold provided by its sister to migrate away from the ventricular zone

  = production of 1 radial glia + 1 neuron/intermediate progenitor

what does it mean that radial glia cells are stem cells?

• they can either self-renew = give more of themself

• they can give rise to other cell types

what are the 2 determining factors that determine whether a cell will give rise to 2 radial glial cells or 1 radial glial + 1 neuron?

1. cell division plane

2. Notch signalling

explain cell division as a determining factor

1. division along meridian (north to south) = generates 2 identical daughter cells (identical to mother)

2. division along equator = generates 2 different daughter cells (asymmetrical localisation) = 1 identical to mother + 1 different from mother/sister

explain Notch signalling as a determining factor

• asymmetric cell division creates daughter cells with different levels of Notch/Delta

• high Notch activation → cell becomes a supporting/progenitor cell

• high Delta expression → cell differentiates into a neuron

• neuronal precursors inhibit Notch in their neighbours via Delta → ensures balanced neuron vs. progenitor numbers

how does lateral inhibition work?

1. 2 cells start equally capable of making and receiving an inhibitory signal

2. a change/bias is introduced - as a result 1 of the cells begins to make more inhibitory signal

3. the second cell receives more inhibitory signals and therefore becomes inhibited

4. to stabilise this change - the inhibited cell must be prevented from continuing to send the inhibitory signal

what is Delta-Notch signalling?

• a cell to cell communication system that helps neighbouring cells adopt different fates

• this system ensures only some cells become neuron while others become support cells (glia/progenitor)

• signal = Delta

• receptor = Notch

what is achaete scute proteins?

• transcriptional factors which when expressed at high levels will turn on TFs that tell the cell to become a neuron

explain the steps of Delta-Notch signalling

1. achaete-scute activates Delta in one cell

2. Delta binds to Notch receptor on its neighbouring cell

3. that neighbour’s Notch pathway activates - stopping it from becoming a neuron

4. the cell with high Delta becomes neuron

5. the cell (neighbour) with high Notch becomes support cell

→ it’s a feedback loop - the more one cells commits to Delta - the more it supresses Delta in its neighbour

how do 2 progenitor cells differentiate?

1. two neighbouring progenitor cells start equally

2. one starts to express a bit more Delta

3. this activates Notch in the other cell

4. Notch supresses Delta and proneural genes in the second cell

5. 1st cell becomes neuron (high Delta)

6. 2nd cell becomes support cell (glia/progenitor) (high Notch)

how do initial tiny differences in Delta/Notch become large fate decisions between neurons and glia?

1. all cells have equal Delta and Notch

   → balanced field = all are progenitor-like

2. a small imbalance begins (from random noise/asymmetric division)

3. as result one cell expresses more Delta - sending more signal to activate Notch in the neighbour

4. the neighbour begins to supress its own Delta

5. the imbalance amplifies via feedback

   • high Delta cell supresses Notch = becomes a neuron

   • high Notch cell supresses proneural genes = becomes a support cell

what happens if Notch signalling is activated?

• the cell becomes a support cell = remains progenitor

what happens if Notch signalling is inhibited?

• the cell becomes a neuron

how was the Notch pathway discovered in Drosophila?

• researchers studies how cells in the developing neurogenic region decide whether to become neurons (neuroblasts) or not

what is the neurogenic region?

• the part that gives rise to the nervous system (NS)

what does the wildtype Drosophila (normal) express?

• many clusters of cells in the neurogenic region express proneural genes like achaete and scute

• these cells are able to become neurons

• but through lateral inhibition only a few become neurons

• the rest become support cells

what was observed in the proneural gene mutant - (achaete-scute mutant)?

• the achaete/scute genes are needed to start the process of neuronal formation

• if these genes are knocked out = no neurons form

• no achaete scute = no neurons form

what was observed in the neurogenic mutant (Notch pathway mutant)?

• if Notch is knocked out = cell can’t receive the inhibition signal

• all of the cells become neurons

• as result too many neurons form

• showing that Notch normally inhibits some cells from becoming neurons

CLINICAL WORK

1. Understanding of congenital conditions (through the understanding of signals and TFs that build the NS)

• Shh is expressed in floor-plate cells in the forebrain and spinal cord

• in Shh knock out mice (Shh -/-) = the mice develop but they show holoprosencephaly and cyclopia

what is holoprosencephaly?

• occurs when the 2 sides of the brain don’t sit as separate sides but fuse into 1

what is cyclopia?

• there’s no floor plate = no eye field separating into 2 = so as result it’s 1 single eye

CLINICAL WORK

2. Importance in drug discovery - disease understanding and regenerative medicine

• motor neuron disease

• through studies in mouse/chick/zebra fish

  —> we learned that neural plate is posteriorized through Wnt (+ retinoic acid signals) and its centralised through Shh

• apply BMP antagonists to mouse (ES cells) to get them to neural identity

• Apply Wnt + retinoic acid to get them to posterior identity

• Apply Shh to get them to ventral neural progenitors

what does Shh expression governs ventralisation along?

• along the entire rosto-caudal axis

• due to intersection of Shh with factors that govern A-P regionalisation - different types of neurons are both at the same level along the A-P axis

what are the 3 types of neurons born at the same level along the A-P axis?

1. hypothalamic neurons

2. Midbrain DA neurons (degenerative in Parkinson’s disease)

3. Hindbrain serotonergic neurons ( poor function in depression)

what is the induced pluripotent stem cells (IPS cells) program?

• a 2 step programme to support in vitro differentiation of human cells

• Step 1 = take a differentiated cell from the body and genetically re-programme it into a pluripotent stem cell fate (induced)

• Step 2 = culture IPS cells with factors that control differentiation in the embryo - to obtain a dish of all types of specialised cells

what are the advantages of IPS?

• no need for an embryo

• can get individual specific pluripotent cells

• ability to differentiate inti multiple cell types

• vastly renewable

• easily accessible

• individual-specific (personalised/non-immunogenic)

what are gastruloids?

• 3D aggregates of embryonic stem cells that recapitulate the axial organisation of post-implantation embryos

explain the generation of gastruloids

• human gastruloids can be generated from human induced pluripotent stem cells

• Wnt inhibition can promote some aspects of brain development in gastruloids of some species but not in human

• gastruloids from some species can develop beating hearts