Corticogenesis

John Kerk and Felicity Gore

Stem cell  Neural Progenitor Cell 
Brain

 Neural ‘Induction’

Anterior
- Brain
Dorsal
 Anterior-Posterior Patterning

Left Right

 Dorsal-Ventral Patterning Ventral

Posterior
– Spinal
Cord
 Neural Induction

 Spemann and Mangold, 1924

 Conclusion: there must be a dorsally secreted

‘neural induction’ factor. So began the search...
 65 years later…
 Neural is the default for ectoderm.

Animal Pole

Vegetal Pole
Grunz and Tacke, 1989

 Largely ignored – confirmed by dominant negative activin

receptor studies.
Default model of neural development

Noggin, Chordin,
Chordin
Follistatin

BMP4

P
P
BMP
Epidermis Neural
4
Neural Tube
Anterior-Posterior Patterning

RA

Wnt FGF
 Dorsal-Ventral Patterning
BMP
 Dorsal
 Spinal Cord – Sensory Neurones
 Brain – Pyramidal Neurones
(Ventricular zone)

 Ventral
 Spinal Cord – Motor Neurons
 Brain – Inhibitory Neurones
(Ganglionic Eminances) Shh
 Corticogenesis

Excitatory cerebral neurons develop from

dorsally positioned neural precursor cells

Cerebral neurons are specified according to:

1) Lamina  connectivity
2) Area  functional ID

How does this lamina and areal ID arise?

 Laminar Fate

Cortex is a 6 layered structure.

Layer 1 = Cajal-Retzius cells, 1st

born, produce Reelin – guides later born
cell fates.

Following layer 1 cortex formed in

inside-out fashion 65432

What dictates this order?

 Laminar fate specification in
Drosophila
The Drosophila nervous system develops from
Neuroblasts.
NB

GMC

Neuron

With every division a new GMC is produced.

 Laminar fate specification in
Drosophila

Every GMC encodes a different lamina

daughter cell, with the most superficial layers
produced first and the deepest layers born
last.
1st born GMC A
2nd born GMC B
3rd born GMC C

NB
 How is this regulated?

Intrinsic or extrinsic?
A
Laser ablation studies
A
A B
A A C
New NB

Cell order must be intrinsically regulated

 Intrinsic Program

Is there an intrinsic program within the

Neuroblast or does the sequential cell
production depend on feedback from the cell
being produced?

A B
 Laminar fate in Drosophila is
temporally specified

Cell layer fate depends on a temporal program

intrinsic to the neuroblast.

With every division the neuroblast switches to the

next cell fate.

These cell fates are governed by transcription

factors. Overexpression and knockout leads to an
increase and reduction of lamina respectively, e.g.
Hb, Kr
Relation to vertebrate corticogenesis?

Cortex is laminar.

Sequential birth of neurons

Walsh and Cepko, 1988 – retroviral lineage
analysis
Birthdating studies

But does this mean there is the same

underlying mechanism as in invertebrates?
 Intrinsic factors

Sally Temple’s Clonal Analysis – Shen et al., 06

Single NPCs dissociated and cultured to reveal
the intrinsic components of neurogenesis.

Immunohistochemistry – single NPCs pass layer

165432 suggesting intrinsic
mechanisms at play akin to Drosophila.

Hb homologues have been found.

 But... McConnell and Kaznowski, 91

Labelled proliferating cortical cells with 3H-Thymidine and transplanted

these labelled cells into the cortex of a younger or older ferret.

Phase of cell Layer donor Layer host Result

cycle forming forming
G2 2/3 6 2/3
G2 6 2/3 6
S 2/3 6 2/3
S 6 2/3 2/3

Early born progenitors (S phase, deep layer) will adopt later cell fates, but
not vice versa.

Vertebrate corticogenesis appears to have intrinsic and extrinsic

components.
 Resolution?

FoxG1 – transcription factor expressed by all layers except the

Cajal Retzius cells.

Hanashima et al., 2004

FoxG1 deactivation at E13 results
in reinitiation of Cajal Retzius cell
production.

Intrinsic = progressive shutting off of cell fates to cease early cell

fate production
Extrinsic = factors produced to “pull” cells to more superficial
fates.
An intrinsic mechanism of corticogenesis
from embryonic stem cells

Gaspard et al., 2008

Question: Can an embryonic stem cell produce a

functional neuron in vitro?
 Methods

Adherant monoculture of embryonic stem cells

In contrast to conventional embroid body protocol

Addition of cyclopamine (inhibitor of Shh) to

drive dorsal, and hence pyramidal, neuronal
cell fate.

Transplantation into neonatal mice

Result 1: Pyramidal neurones
successfully made
… these neurons electrically resemble
pyramidal cells
… and morphologically
Sequential neurogenesis displayed

In vivo laminar Expression

expression onset
Pulse chase
Confirmation by clonal analyses
Highly suggestive of ‘an intrinsic mechanism of
corticogenesis from embryonic stem cells’

BUT…

There is a huge under-

representation of late
born cell fates. Could
this represent the lack of
an extrinsic factor
drawing cells out to their
maximal competency?
 Transplantation

Transplant neurons differentiated from TAU –GFP knock in line

for different durations into neonatal mice cortex (frontal).

Significant differentiation duration dependent projections

Short  thalamic projections (presumptive layer 6)
Intermediate  midbrain projections (presumptive layer 5)
Long  cortical projections (presumptive layer 2/3)

 Sequential neurogenesis therefore seems to have functional,

or at least wiring, significance.
 And…

Found robust cortico-cortical projections – i.e. from layer 2/3

suggesting an in vivo factor could indeed underlie the under-
representation of upper layers.
 Areal

Areal projections largely to visual cortex and

thalamic nuclei, as well as limbic areas

Occurred even at short differentiation duration

Correlated to pre-transplantation expression of

COUPTFI and COUPTFII indicative of being a
product of the differentiation protocol

BUT no control used

 Conclusions

This work has largely confirmed ideas surrounding the

temporal intrinsic regulation of laminar corticogenesis as
well as reinforcing the concept of an extrinsic signal dictating
outer lamina fates

So, can an embryonic stem cell produce a functional pyramidal

neuron in vitro? Yes

However the areal result seen in the absence of a control has

raised further questions regarding the prespecification of
areal fate