Tuesday:

Monday:
Induced pluripotent stem
Neural Development
technology
The magic of the biology
The magic of applying our
behind the creation of the
knowledge to create the
human brain.
human brain in vitro (models
Development of the
nervous system
Sebastian Illes
Department of Physiology
Institute for Neuroscience and Physiology
Contact: sebastian.illes@neuro.gu.se

February, 2023
The Neural development lecture and further information
• Why the human (brain) development is a fascinating process?
• Early developmental process: From fertilization to blastula/blastocyst
• Gastrulation
• Neurulation / Neural induction
• Patterning (early stages of brain region specification)
• Embryonic neurogenesis and gliogenesis

Above listed sections are linked to the slides within this power point presentation. Each
slide has in the right upper corner an ”overview” box that is linked to this overview slide.
I will provide references where you can inform yourself about other neural developmental
process taht are not covered in this lecture, e.g. axonal growth, synapse development,
neural cell death, electrophysiological maturation. (optional)
Exam questions can be answered solely based on the information the slides provide.
Slides will be uploaded after the lectures have been held.
 overview
Why the human (brain) development is a fascinating process?
Human brain
Blastocyst Volume around 1180-1260 cm3, measures about 5.5 x 6.5 x 3.6
• Human: 5th day of development (5 days post- inches (140 x 167 x 93 mm). It weighs about 3 pounds (1.3 kg).
conception). 86.000.000.000 neurons + 5 to 10x 86.000.000.000 glia cells
• 24 up to 322 cells, = 516 000 000 000 to 946 000 000 000 cells
• size is about 0.2mm
• Comprise of
• the trophectoderm
• the inner cell mass
• the blastocoel.
 overview
From fertilization to morula towards blastocyst

Fertilization: A process where the haploid

gametes, the spermatozoa and the
oocyte, fuse to give rise to diploid zygote.

Morula: A mass of blastomeres, which

arise from zygote, fertilized oocyte, by a
number of cleavages. Its name derives
from its resemblance to a mulberry (Latin:
morum).

Cleavages: a series of rapid cell divisions

(mitosis)

Blastomere: Cell produced by mitosis

after fertilization and many blastomeres
https://www.conceptfertility.co.uk/fertility-treatments/ivf-treatment/blastocyst-culture-and-transfer/ build the morula and blastula/blastocyst.
 overview
Blastocyst development
 overview
Def. cell lineage and cleavages
Cell lineage: The developmental history of a tissue or
organ from the fertilized embryo.
Cell lineage analysis is tracking of organism's cellular
ancestry due to the cell divisions and relocation as time
progresses, this starts with the originator cells and
finishing with a mature cell that can no longer divide.

C. elegans nematode
form has only 959
cells, the enterie
development of the
the worm takes only
2 days.

Caenorhabditis
https://www.youtube.com/watch?v=M2ApXHhYbaw elegans
 overview
Gastrulation in worm and fly
Caenorhabditis elegans Drosophila melanogaster
 overview
Gastrulation in frog
• Gastrulation: Process, which
occurs in all animals, where
blastomeres become
specified and spatial
reorganise into the three
germ-layers: endo-, meso-
and ectoderm. The cellular
movements of the cells is
different between animals.
The movements and
progressive specification of
cells has created new
“tissue” relationships.
• Gastrula: Multicellular,
transient, developmental
structure comprise of the
three germ-layers: endo-,
meso- and ectoderm.
 overview
Key experiments that helped to develop the concept of a
“neural inducer”.
 overview
Key experiments that helped to develop the concept of a
“neural inducer”.
Spemann-Mangold experiment
Mangold started PhD in spring 1921, published 1924,
1935 noble price to Spemann.

Organizer/Inducer: A part of tissue or group of cells that instruct

neighbouring cells to adopt a developmental fate and instruct
the cells to develop into a specific tissue or part of the body.
 overview
Key experiments to answer: How does an inducer work?
Which are the neural inducing factors?
• 1970-1985:
 overview
Discovery of the first neural inducing factor (1992, Harland and Smith)
A.Normal development of a Xenopus embryo.
B. UV light treatment of the early embryo inhibits the development of
dorsal structures by disrupting the cytoskeleton rearrangements that
pattern the dorsal inducing molecules prior to gastrulation
C. Lithium treatment of the early embryo has the opposite effect; the
embryo develops more than normal dorsal tissue.
D.If messenger RNA is extracted from the hyperdorsalized embryos and
injected into a UV-treated embryo, the messages encoded in the
mRNA can "rescue" the UV-treated embryo and it develops relatively
normally.
E. Similarly, cDNA from the organizer region of a normal embryo can
rescue a UV-treated embryo. The Noggin gene was isolated as a
cDNA from the organizer region that could rescue the UV-treated
embryo when injected into the embryo.
F. Recombinant protein was made from this cDNA and shown to induce
(F) neural tissue from isolated animal caps, without any induction of
mesodermal genes.
 overview
BMP, TGF-beta and FGF signalling in neural induction
 overview
Neurulation in frog
• Neurulation: Process,
which occurs in all animals,
where neuro-ectoderm
cells of the neural plate
invaginates and form the
neural tube.
• Neurula: Multicellular,
transient, developmental
structure comprise of the
three germ-layers: endo-,
meso-, ectoderm and
neuroectoderm (neural
plate).
 overview
Neural tube formation
 overview
From Zygote to gastrula towards neurula in human
• https://www.youtube.com/watch?v=UgT5rUQ9EmQ
 overview
After forming neural tube:
patterning of the brain and spinal cord
 overview

Terms related to patterning

Cleavages: a series of rapid cell divisions

• Anterior: In front of; toward the face
• Posterior: Behind; toward the back
• Superior: Above; toward the head
• Inferior: Below; toward the feet
• Medial: Toward the middle
• Lateral: Toward the edge
• Dorsal: Toward the top of the brain or the back https://openbooks.lib.msu.edu/neuroscience/chapter/anatomical-terminology/
of the spinal cord
• Ventral: Toward the bottom of the brain or the
front of the spinal cord
• Rostral: Toward the front of the brain or the top
of the spinal cord
• Caudal: Toward the back of the brain or the
bottom of the spinal cord
 overview
Segmentation genes
• The unique positional identity of the segments in Drosophila is
derived by a program of molecular steps, each of which
progressively subdivides the embryo into smaller and smaller
domains of expression.
• The oocyte has two opposing gradients of mRNA for the
maternal effect genes; bicoid and hunchback are localized to
the anterior half, while caudal and nanos messages are
localized to the posterior regions.
• The maternal effect gene products regulate the expression of
the gap genes, the next set of key transcriptional regulators,
which are more spatially restricted in their expression.
• Specific combinations of the gap gene products in turn activate
the transcription of the pair-rule genes, each of which is only
expressed in a region of the embryo about two segments wide.
• The pair-rule gene together with a second set of periodically
expressed genes, the segment polarity genes….
• …determine the specific expression pattern of the homeotic
genes. In this way, each segment develops a unique identity.
 overview
Sequential chromosomal homeobox (hox)-genes
• Studies of Hox genes in neural
development have concentrated on the
hindbrain.
• Hox gene clusters in arthropods
(Drosophila) and vertebrates (mouse
embryo) have a similar spatial organization
and similar order along the chromosomes.
• In Drosophila, the Hox gene cluster is
aligned on the chromosome such that the
anteriormost expressed gene is 3′ and the
posterior most gene is 5′.
• In the mouse, there are four separate Hox
gene clusters on four different
chromosomes, but the overall order is
similar to that in arthropods: the anterior
to posterior order of gene expression is in
a 3′–5′ order on the chromosomes.
 overview
Rhombomeres and motorneurons
• Rhombomeres are repeated
morphological subdivisions of the
hindbrain.
• The rhombomeres are numbered
from the anterior most unit, r1,
just posterior to the midbrain
(mesencephalon), to the
posterior most unit, r8, at the
junction of the hindbrain with
the spinal cord.
• The motor neurons send their
axons through defined points at
alternating rhombomeres.
• The members of the Hox gene
cluster are expressed in a 3′–5′
order in the rhombomeres.
 overview
Effects of rhombomere development in hox-gene KO-mice
Rhombomere identity is determined by the Hox code. Hox gene knockouts in mice affect the
development of specific rhombomeres.

Wild-type animals have a stereotypic pattern of motor
neurons in the hindbrain. The trigeminal (V) cranial
nerve motor neurons are generated from r2 and r3,
while the facial nerve motor neurons are produced in r4
and the abducens motor neurons are produced by r5.
Deletion of the Hoxa1 gene in mice causes the
complete loss of rhombomere 5 and a reduction of
rhombomere 4 (rx). The abducens motor neurons
are lost in the knockout animals, and the number of
facial motor neurons is reduced.
 overview
Segmentation genes for forebrain development
 overview
Rostro-caudal morphogen gradients

Wnt (A) and Fg/8 (B) form an interconnected network

that specifies this boundary and is necessary for the
growth of the midbrain and the cerebellum. Deletion of
any of these molecules in mice results in a loss of the
midbrain and reduction in cerebellar size.

C: A wild-type embryo is shown in C. D:

Wnt/ knockout mouse brain is shown Note
the loss of the midbrain (mb) and
cerebellum (cb) in the mutant brain. Other
structures are normal (T. telencephalon; di.
diencephalon; my, myelencephalon; sc.
spinal cord; tb. tailbud).
 overview
Dorso-ventral polarity and segmentation
 overview
Signal specify ventral fate in developing spinal cord
 overview
Signal specify dorsal fate in developing spinal cord
 overview
Dorso-ventro morphogen gradients
 overview
Wnt signalling
• In Wnts are secreted molecules, but they are typically
associated with the cell membrane and diffuse only limited
distances from the cell that secretes them.
• Wnt proteins bind to a receptor called Frizzled.
• Along with the Frizzled receptor, there is a second
component to the receptor complex, the L RP protein.
• In the absence of Wnt, an intracellular protein, ß-catenin,
associates with several other proteins, including Axin,
GSK3ß, and APC. This complex is continually degraded, and
so exists only transiently in the cell.
• when Wnt binds to Frizzled, another protein called
Disheveled blocks the degradation of the complex and
causes the ß-catenin to accumulate.
• As the ß-catenin accumulates, some of it moves to the
nucleus, where it forms a complex with a different protein,
TCF.
• The ß-catenin/TCF complex can bind to DNA at specific
sequences and activate target genes
 overview
Anterior and posterior patterning of the cortex

Transcription factor
Morphogen gradients
expression profile
 overview

• Neuronal and glial diversity

• Cytoarchitecture of brain regions
 overview
Birth dating studies demonstrate the sequential neurogenesis
and inside-out pattern of cerebral cortical development.
 overview
Neural stem cell properties: Interkinetic nuclear migration
 overview
Basic molecular mechanisms of the mitotic cell cycle
• Mitogens (molecule that promote cell
proliferation) are shown in green and those
that inhibit the cell cycle are shown in red.
• The entry of a cell into S-phase is one of the
key check points on mitosis.
• E2f1 and CycE/Cdk2 complexes cause cells
to enter S-phase.
• the Rb protein and cyclin-dependent kinase
inhibitors, like p27 kip, act like “brakes” on
S-phase entry.
• Mitogens that stimulate cells to enter the
cell cycle, like EGF and FGF, stimulate CycD
expression or stabilization, which then
inhibits the “brakes” on the system and
promotes S-phase.
 overview
Temporal shift from proliferation to more differentiation
• P=proliferative fraction Neural stem cell
• Q=quit fraction neuron
 overview
Radial glial cells and basal progenitor cells
 overview
Mechanism: Proliferative cells are polarized leading to
asymmetric distribution of RNA and proteins in dividing cells
 overview
Mechanism: Genes are expressed in oscillatory cycles.
 overview
Sequential geneses of neuronal subtypes
 overview
Inside-out cortical development by migration
 overview
Radial and tangential migration
 overview
Neural cell lineage in vitro analysis (neuro to glial switch)
 overview
In vivo time-dependent neuro to glial switch
 overview
Factors regulating neuronal and astrocyte development
 overview
References and further readings
Developmental wiki:
• https://embryo.asu.edu/pages/spe
mann-mangold-organizer

Some links to videos showing the

magic from zyogote to living animals:
• https://www.youtube.com/watch?v
=SEejivHRIbE
• https://www.youtube.com/watch?v
=M2ApXHhYbaw
• https://www.youtube.com/watch?v
=uCn1PQP2yAo