Week 1 Topic 1 - Overview of CNS development

Question 1

What two levels do we study that neural development takes place?

Answer 1

We can think of neural development as taking place on two levels, namely, a ‘systems’ level and a
cellular level.

Question 2

What does a systems level approach consider?

Answer 2

In this topic, we will first consider the systems level, looking at the

1. changes in size and
2. changes in shape that occur during the embryonic development of the nervous system.

This process is called
morphogenesis.

Question 3

What is morphogenesis?

Answer 3

changes in shape that occur during the embryonic development of the nervous system.

Question 4

What do changes at the cellular level include? What is the developmental process and what is it called?

Answer 4

Next, we will describe changes at the cellular level that allow cells to change from dividing progenitors into mature neurons with complex morphologies, interconnected in circuits. This process is called differentiation.

Question 5

What are the 10 Levels of Neural Development?

Answer 5

1. In humans, development begins with fertilisation of the egg, which cleaves to give rise to a ball of cells
   called a blastocyst.
2. Implantation of the embryo into the uterine wall occurs at the end of the first
   week.
3. And further development generates a two-layered embryonic disc, consisting of hypoblast and
   an epiblast.
4. At the end of the second week, the process of gastrulation transforms this disc into a three-layered structure consisting of three so-called ‘germ’ layers– the ectoderm, mesoderm, and endoderm– which give rise to all the tissues of the body.
5. By the end of the third week, the process of neurulation begins, which creates the embryonic
   nervous system.
6. Amazingly, in weeks 4-5, the embryo starts to be recognisable, with a head, tail, and
   some of the embryonic structures that will be present in the adult, such as the limb buds, which grow
   into the limbs. This stage is often called the ‘tailbud’ stage.
7. In humans, the second month of gestation is referred to as the embryonic period, during which the
   major organ systems start to form.
8. And months three to nine is the foetal period, which is mainly
   concerned with growth. Large amounts of cell proliferation take place, a process which is particularly
   important for the brain.
9. Further development of the nervous system involves the ectoderm, which develops under the
   influence of signals from the underlying mesoderm, a process called neural induction. During this
   process, a portion of the ectodermal germ layer is induced to become neural tissue, which will form
   the nervous system.
10. As the tissue becomes neural, it also undergoes morphogenetic changes in shape, called neurulation.

Question 6

Describe the process of neurulation within the context of embryotic development. This is a long 35 step process.

Answer 6

We will now look at neurulation in more detail, visualising the process of neurulation in a surface view, a transverse view, and in scanning electron micrographs of a human embryo.

1. Early neurulation, at three weeks.
2. The surface view is shown with the anterior, or cranial, end of the
   embryo at the top of the picture and the posterior, or caudal, end at the bottom of the picture.
3. The arrow shows the level of the surface view at which the transverse section is taken.
4. In the transverse section, at 19 and 20 days, we can see that the embryo consists of three germ layers.
5. The ectoderm lies on top, and the medial part will give rise to the nervous system, with the more lateral regions giving rise to the epidermis of the skin.
6. The medial part forms the neural plate, which has started to thicken.
7. Underneath it lies the mesoderm, consisting of the notochord, medially, and two other blocks of mesoderm, laterally. Underneath this lies the endoderm.
8. We can see that between 19 and 20 days, the neural folds rise up on either side of the midline and form a v-shape.
9. Somites form from some of the mesoderm underneath these folds, which will later form the axial muscles.
10. In the surface view, the somites can also be seen to form small blocks of
    tissue, and the embryonic disc to lengthen further. In the surface view, we can see how the neural folds form first at one axial level.
11. In the scanning electron micrograph of the human embryo, the neural tube looks somewhat striated because of the somite blocks beneath it.
12. During later neurulation, at 22 to 23 days, looking first at the transverse section, the neural folds can be seen to approach each other, and the somites to have expanded.
13. Eventually, the neural tube closes and becomes enclosed and separated from the layer of ectoderm which forms over the top.
14. In the surface view, it is clear that one region of the neural tube has started to fuse.
15. This is typically the neck region.
16. At the later stage we can see that the neural tube starts to zip up, towards the anterior and the posterior ends, and the nervous system starts to be subdivided, with the spinal cord posteriorly, and the brain vesicles, or subdivisions, more anteriorly.
17. The scanning electron micrograph of the human embryo is very similar to the diagram, but the embryo is attached to extra embryonic membranes which will later cover it.
18. By the tailbud stage, which is at four to five weeks of gestation, cranial and caudal folding has occurred, arching the body to give it what has sometimes been referred to as a ‘comma’ shape.
19. Lateral folding has also occurred to enclose all forming internal organs in a covering of ectoderm, which will become the skin.
20. We can see that the embryo has acquired a more recognisable
    appearance, with a head and tail somite blocks, and structures called branchial, or pharyngeal, arches, which will form elements of the lower jaw and neck.
21. At a later stage of human development shown, you can also see the limb buds, outpocketings of tissue which will eventually grow into the limbs.
22. The diagram highlights the developing eye, and the
    optic vesicle, which will give rise to the inner ear. In this way, the major structures of the developing
    embryo are already formed by four to five weeks.
23. Surprisingly, the embryo of a human looks extremely similar to that of other animal groups at this stage.
24. These beautiful drawings by the 19th century embryologist, Haeckel, show that at the tailbud
    stage, all the essential features of the body plan are present and look similar, even though the
    eventual body plan of these different organisms is rather different.
25. Haeckel may have exaggerated the similarities somewhat, for effect, but the basic idea is correct.
26. This also brings home the point of why we can study a variety of different experimental organisms in
    order to understand more about human development.
27. Looking now at the further development of the neural tube without the other tissues, we can see that the cranial to caudal folding of the tube has taken place in concert with the folding of the rest of the embryo.
28. Several subdivisions now appear in the tube.
29. Whereas the region of the developing
    spinal cord remains with a small diameter, the forebrain, midbrain, and hindbrain, also termed the prosencephalon, mesencephalon, and rhombencephalon, have started to expand.
30. The prosencephalon is divided into the telencephalon more cranially, and the diencephalon more caudally.
31. The telencephalon is later destined to give rise to most of the cerebral hemispheres via an extensive folding process.
32. The diencephalon will give rise to some of the important collections of
    neurons, such as the thalamus.
33. These sorts of collections of neurons are termed nuclei.
34. The folding of the neural tube which occurs involves the formation of flexures. In this way, convex flexures at the midbrain, and at the level of the junction between the spinal cord and the hindbrain, create the more mature morphology.
35. A third, concave flexure also appears in the hindbrain, which
    means that the cranial part of the hindbrain, called the pons, becomes separated from the more caudal region, the medulla.

Question 7

What is neurulation?

Answer 7

Neurulation refers to the folding process in vertebrate embryos, which includes the transformation of the neural plate into the neural tube

Question 8

What is differentiation?

Answer 8

During development, individual cells go through a process of differentiation. We can broadly think of development as a process where cells progress from a ‘multipotent’ population, capable of producing a range of cellular derivatives, to cells of particular, specialised identities, or ‘fates’.

Question 9

What is the Waddington representation?

Answer 9

The embryologist, Waddington, nicely represented this as a ball rolling down a hill– the epigenetic
landscape– and then rolling into one of a number of channels.

Which channel the ball ends up in is not random, however.

It depends on a number of events which take place in development, especially external influences and interactions between groups of cells, which instruct cells on their next developmental step.

The process of neural induction is an example of this, in which the neural plate is influenced by the mesoderm to develop into the nervous system.

We can thus see cell differentiation as a decision tree, which will eventually lead to cells assuming one of a number of fates.

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Question 10

What is neural induction?

Answer 10

neural induction is defined as the step when ectodermal cells become ‘specified’ as neural stem or precursor cells (Table 1). Later in development, these specified cells will no longer respond to signals that induce alternative fates, and have thus ‘committed’ to a neural fate

Question 11

What are the four aspects of neuronal differentiation?

Answer 11

There are various different aspects of neuronal differentiation.

1. One is the appearance, or morphology, of individual cells, shown here using the examples of a Purkinje neuron and a pyramidal
   neuron.

The Purkinje neuron resides in the cerebellum and has an extremely elaborate dendritic tree, whereas the pyramidal neuron resides in the cerebral cortex and is less elaborate, with an apical dendrite and some branches. Both neurons have an axon which extends downwards in the diagram, exiting the cerebellum or cortex to project to other parts of the brain.

Other aspects of neuronal differentiation are

2. the gene expression profile,
3. neurotransmitter type, and
4. connectivity to other neurons in the nervous system.

Together, all of these features make up the individual characteristic of differentiated neuronal types.

Question 12

What are the 5 developmental steps which lead to differentiation?

Answer 12

1. neurogenesis, during which cell division occurs to generate neurons;
2. cell migration, when young
   neurons migrate away from the ventricular zone;
3. axonogenesis, when the neuron starts to develop
   processes, including an axon which grows out towards targets;
4. synaptogensis, when axons make
   contact with their target neurons or other structures;
5. cell death or pruning, when regressive events
   often occur, leading to the formation of the mature neuron.

Question 13

Describe the 7-step process of embryonic neurogenesis.

Answer 13

1. The neural tube is divided into a ventricular zone, adjacent to the ventricle, which contains the cerebrospinal fluid, and a mantle zone, adjacent to the pial surface covered by the meninges.
2. Radial glial cells are elongated cells with a long process or endfoot on each surface.
3. These are the progenitor cells of the nervous system.
4. Radial glial cells undergo cell divisions repeatedly to expand the progenitor cell population, and some of these divisions give rise to a neuron shown by the shaded cell in the cartoon.
5. The cell divisions themselves, of the cell body, occur adjacent to the ventricular surface of the neurepithelium.
6. Once the neuron is generated in such a cell division, it will migrate along the radial glial cell, using it as
   a guide towards the mantle zone.
7. There, further differentiation of the neuron will take place, including
   extension of an axon.

Question 14

Describe the process of migration and the two types of migration.

Answer 14

1. Neurons frequently have to migrate long distances towards their final position in the developing
   nervous system.

There are two main types of migration–

a. radial migration and
b. tangential migration.

We have already dealt with one type of migration in the previous section using the example of the
spinal cord, in which progenitors of cells migrate radially from the inside to the outside of the neural
tube to generate neurons.

This type of radial migration also occurs in the telencephalon, or forebrain, and is shown here
in a transverse section of the developing telencephalon in a mouse, which will later form the
cerebral hemispheres.

Cells which migrate radially, along radial glia, give rise predominantly to
the neurons with long axons that project to other regions of the nervous system, and that use the
neurotransmitter glutamate, called excitatory projection neurons.

The other type of migration which occurs in the telencephalon is called a tangential migration, in
which neurons migrate orthogonal to the radial axis.

Neuronal progenitors migrate from the ventral
telencephalon into the dorsal telencephalon, the developing cerebral cortex, and intermingle with the
neurons which have undergone radial migrations.

These neurons, which have migrated tangentially,
give rise to neurons with short axons, which use the neurotransmitter GABA, called inhibitory
interneurons.

Our third example of a neuronal migration concerns cells that split off from the ectoderm while
neurulation is underway.

These are the neural crest cells.

Shown in a transverse section of the
developing spinal cord, neural crest cells migrate away from the forming neural tube to form
elements of the peripheral nervous system.

In particular, these are the dorsal root ganglia and
sympathetic ganglia in the trunk, and the cranial ganglia of the head.

Question 15

Describe the process of differentiation and 5 stages of axongenesis.

Answer 15

As neurons move into the mantle zone and start to differentiate, they start to develop an axon.

This process of axonogenesis can be beautifully visualised in hippocampal neurons growing in vitro, as shown by this example from the lab of Gary Banker.

Here you see the different identified stages of axonogenesis and neural development at the single cell level.

At Stage 1, neurons are initially round blobs.

At Stage 2, neurons look radially symmetrical, with
several neurites, or processes.

At Stage 3, one of these neurites becomes selected as an axon in a
process of symmetry breaking. This axon will go on to grow out and extend towards its targets.

At Stage 4, the axon continues to grow and the dendrites start to grow out from the cell body.

At Stage 5, the dendritic tree becomes more elaborate with small protrusions, or dendritic spines, forming on the dendrites. In vitro, the neurons can be seen to form a network.

Question 16

Describe the development of the axon and the dendrites

Answer 16

Development of the axon and the dendrites proceed in parallel.

Growing axons are guided by molecules in their environment to their targets.

Axons eventually make contact with their targets, whether that is a neuron, as in this case, a gland, or a muscle.

In the case of neuron-neuron synapses, these are most frequently made on dendrites, and in fact, the dendritic spines.

Synapses are sometimes made on the neuronal cell body itself– axosomatic– or on an axon– axoaxonic.

Just to make this clear, in the diagram, the synapse is formed by the growing tip of the neuron to the left on the neuron on the right.

Thus, the neuron to the left is the presynaptic part, and the neuron to the right, the postsynaptic part.

Question 17

What are the 7 stages of synaptogenesis?

Answer 17

Once the growth cone has reached its target cell, synapse formation is initiated between the presynaptic axon and the postsynaptic dendrite, soma, or axon.

There are many stages to synaptogenesis.

1. Various molecules, including cell adhesion molecules,
   contact dependent, and diffusible molecules play a role in synapse formation.
2. Neuroligins and neurexins are families of transmembrane proteins that are expressed by the postsynaptic and the presynaptic neuron, respectively, and that are important in the process of synaptogenesis.
3. They bind the pre- and postsynaptic parts of the synapse together, and serve as a focus for other proteins to cluster together to form the synapse.
4. Different members of the neuroligin group are enriched in excitatory and inhibitory synapses, and are involved in specifying these different synaptic types.
5. Molecules such as cadherins and SynCAMs help to consolidate synapse formation.
6. The neurexins and neuroligins then help to recruit specialised groups of proteins into the presynaptic active zones, containing the neurotransmitter vesicles in the presynaptic terminal.
7. They also coordinate the assembly of the postsynaptic densities, which contains so-called scaffolding proteins and neurotransmitter receptors.

Overall, the process of synaptogenesis is a very complex and coordinated process.

Question 18

Describe the pruning process.

Answer 18

The nervous system forms not only by growth and elaboration of axons and dendrites, but also by
sculpting of neuronal architecture and by cell death.

Cell death is a surprisingly common phenomenon
in the nervous system.

It’s estimated that around 50 per cent of motor neurons, for example, die
during later development.

These regressive events may thus involve either the elimination of whole cells, or parts of cells,
axons, synapses, or dendrites. In the examples shown of the developing cortex from humans, the
complexity of the brain can initially be seen to increase, in terms of the density and numbers of
neurons, up to two years of age, with increasing synapse formation.

From four years to six years, however, a process of synapse pruning and consolidation takes place,
and some decrease in the complexity of the brain landscape occurs.

Pruning can occur to axons
and to dendrites, which disintegrate and the debris is then cleared away.

Cell death and pruning may
eliminate unwanted neurons or connections, match numbers of pre- and postsynaptic cells, and
ensure that synaptic transmission and circuit function is optimised.

It’s not completely clear why these events occur, but it may be to ensure that there are matching
numbers of pre- and postsynaptic cells.

In addition, the removal of any aberrant or unwanted
connections may occur, and the fidelity of connections in terms of structure and function may be
improved.

Question 19

What percentage of motor neurons die during later development?

Answer 19

It’s estimated that around 50 per cent of motor neurons, for example, die
during later development.

Question 20

What are some examples of human disorders which can be caused by defective developmental processes?

Answer 20

Examples of human disorders which can be caused by defective developmental processes are

1. autistic spectrum disorder,
2. schizophrenia,
3. childhood onset epilepsy, and
4. X-linked mental retardation.

Question 21

neurodevelopmental disorders in humans

some of the genes that are
mutated in humans with these disorders

dendrite and synapse development are often affected
by these gene mutations

Answer 21

In this section, we will highlight certain developmental aspects of ASD and schizophrenia.

Understanding the developmental processes I have described in this subtopic is extremely important,
as this can give insight into neurodevelopmental disorders in humans.

Large-scale human genetic screenings and experiments in animal models have started to uncover
some of the principles that underlie these disorders.

We now know some of the genes that are
mutated in humans with these disorders.

Developmental neuroscientists are trying to understand the mechanisms that underlie the changes caused by these mutations.

It’s emerged in recent years in particular that dendrite and synapse development are often affected
by these gene mutations.

Many aspects of development can be perturbed to lead to such disorders,
such as axon growth, guidance, neuronal migration, synapse formation, and function.

Question 22

ASD is a neurodevelopmental disorder and mutations in several genes including neuroligin-4 are linked to
autistic spectrum disorder.

Answer 22

Autistic spectrum disorder, ASD, is an umbrella term for a disorder which can take on multiple forms
and have multiple causes. Nevertheless, it is clear that ASD is a neurodevelopmental disorder and
that some forms of ASD are genetically based.

In humans, it has been shown that mutations in several genes including neuroligin-4 are linked to
autistic spectrum disorder.

Neuroligin-4 is involved in synapse development. Studies using mice,
which are deficient in the neuroligin-4 gene, called knockout mice, have shown that markers of
inhibitory synapses are reduced in some areas of the hippocampus, pointing to a developmental
defect.

In the figure, you can see the white staining representing immunofluorescence for two markers of
inhibitory synapses– gephyrin and a GABA A receptor subunit. Compared to the wild-type panels to
the left, there’s a reduction in the staining in the neuroligin for KO, or knockout, mouse. In addition,
neuroligin for knockout mice showed behavioural changes reminiscent of ASD.

It’s perhaps surprising that it’s been possible to model some of the behavioural aspects of ASD in
mice using assays of, for example, social interaction or vocalisation.

These experiments have shown
that neuroligin for knockout mice showed impairments in social interaction and communication, as
well as repetitive behaviours and interests. Some of these features are characteristic of humans with
ASD.

Whereas the process of synaptogenesis and its link to behaviour is extremely complex, these
studies give us hope that ASD can be modelled using the mouse as an experimental system.

As well as changes in the numbers of synapses, there may be changes in the structural features
of dendrites and dendritic spines which are key to synapse formation.

There is a large amount of
evidence now to show that the numbers, shape, and development of dendritic spines change in some
individuals with schizophrenia or ASD including X-linked mental retardation, Fragile-X, which has
features of ASD.

The number of dendritic spines is reduced in the dorsolateral prefrontal cortex of
some schizophrenia subjects.

You can see this in the figure where the black line with the blobs on it represents a dendrite and the
dendritic spines in a normal subject.

In the two schizophrenia subjects shown, you can see that the
number of dendritic spines looks to be reduced.

This may reflect defects in either the process of
dendrite development and/or pruning.

Mice lacking the Fragile-X mental retardation protein have more immature, thin spines, and there is
evidence for a similar change in humans with Fragile-X.

You can see this in the figure comparing a
dendrite from the wild-type animal with a mouse used as a model for Fragile-X and lacking the FMRP
protein.
Knowledge about the genes and proteins which are involved in dendritic spine development
and testing their role using animal models will be key to understanding the link between spine
development, function, and mental health.

It’s worth bearing in mind, however, that there are a large
variety of studies on issues such as dendritic spine development, density, and maturation.

Some of
the results from these studies are conflicting, and much further work will be required before we can
draw general conclusions from this work.

We can conclude that understanding development in detail can unlock many of the secrets of the
way the nervous system is built and how it later functions.

Research in developmental neuroscience
will help us understand neurodevelopmental disorders and mental health and vice versa,

Many
of the techniques currently being used and under development will play an essential role in the
next decades in unravelling these principles.

For example, live imaging in vivo using the mouse and
zebrafish can now tell us much about the dynamic events that occur during dendrite development,
pruning, and plasticity.

Behavioural tests in the mouse are also beginning to give us insight into the effect of particular genes
and proteins on behaviour at the organism level.

Genetic screens in humans can then reveal the
genes whose function can be tested in animals, while genes shown to be important in development
through basic science studies can be screened in the human population.

This interplay will be the
foundation of future discoveries in development and mental health.