L21 Nervous System Nervous system development

Overview

This lecture follows the nervous system from a two-layered embryonic disc to a brain that is still maturing in the mid-to-late 20s. It runs in four linked parts: neurulation (how ectoderm becomes the neural tube, and what happens when closure fails), the embryonic origins of the CNS from the neural tube and of the PNS from the neural crest, the building of the 6-layer cerebral cortex by neurogenesis, radial migration and cortical organisation (with the malformations produced when each step fails) plus axon guidance in circuit formation, and finally the timeline of development with the prenatal and postnatal perturbations that derail it.

From fertilisation to the embryonic disc

• A fertilised egg (zygote) progresses zygote -> embryo -> fetus -> baby; human embryonic development occupies the first 8 weeks. Carnegie stages 1-23 chart this, from the 1-day zygote (stage 1) to stage 23 at 54-58 days.
• Cleavage sequence: single cell within the zona pellucida -> 2-cell -> 4-cell -> morula -> free blastocyst (trophoblast, inner cell mass, uterine cavity) -> blastocyst attached to and invading the uterine lining.
• The blastocyst forms at day 5 or 6 after fertilisation and contains 100-200 cells. Human embryos form from its inner cell mass.
• By day 11 after fertilisation the inner cell mass has become the two-layered embryonic disc: embryonic ectoderm (outer) and embryonic endoderm (inner), sitting between the amniotic cavity and the yolk sac.

Neurulation

The entire nervous system arises from embryonic ectoderm. The steps, in order:

1. The ectoderm thickens along the midline axis to form the neural plate (about 19 days).
2. The neural plate invaginates, forming a groove flanked by neural folds (about 20 days). Viewed from the dorsal surface, the embryo shows a central neural groove, neural fold tissue at its border, and surrounding epidermal ectoderm, along an anterior-posterior axis.
3. The neural folds rise and the plate folds inward (about 22 days), and neural crest cells appear at the fold margins.
4. The folds fuse to form the neural tube (about 26 days), which separates from the overlying surface ectoderm.

Fates of the tube and its derivatives:

• Rostral neural tube forms the brain.
• Caudal neural tube forms the spinal cord.
• Cavity of the tube forms the ventricular system.
• Epithelial cells lining the neural tube generate all the neurons of the CNS.
• Neural crest gives rise to neurons destined to reside in ganglia, and to the entire PNS.

Closure times, which are the key to the defects below: the anterior neuropore closes at about 24-26 days, the posterior neuropore at about 26-28 days.

Neural tube defects

Anencephaly (without brain)

• The rostral neural tube fails to fuse.
• The developing brain remains in contact with amniotic fluid, so brain tissue degenerates.
• The hemispheres or the whole forebrain are usually absent.
• Almost always accompanied by severe cleft spinal cord.
• Infants are usually born dead or die soon after birth.
• Occurrence rate about 0.1%.

Spina bifida (forked spine)

The caudal neural tube fails to close. Three types, in increasing severity:

1. Spina bifida occulta (hidden spina bifida). Relatively minor and asymptomatic; often found by accident on an X-ray or similar test. Sometimes a dimple, patch of hair, or red mark at the base of the spine. Affects about 10% of the population.
2. Meningocoele. A sac of spinal fluid pushes through an opening in the spine. The spinal cord and spinal nerves remain in place. Minor disabilities may result.
3. Myelomeningocoele. The spinal canal does not close, so spinal cord and spinal nerves are external. Motor and sensory problems below the sac, plus bowel and bladder problems.

Folate to prevent NTDs

• New Zealand NTD occurrence: about 3.4 per 10,000 live births, plus about 7 per 10,000 non-term pregnancies.
• Folate (folic acid, vitamin B9) is required for DNA metabolism in rapidly dividing cells.
• Found in many fruits (particularly oranges, berries and bananas), leafy green vegetables, cereals and legumes.
• Consumption during pregnancy is associated with reduced rates of NTDs, at 0.4-0.8 mg/day for people who are pregnant or trying to conceive.

Brain vesicles and flexures

Primary vesicles (3)

The rostral-most end of the neural tube expands, because rostral cells divide faster than caudal cells and CSF production is increased anteriorly. By about 26 days there are three vesicles: prosencephalon (forebrain), mesencephalon (midbrain), rhombencephalon (hindbrain), with spinal cord caudally.

Secondary vesicles (5)

Continued differential growth anteriorly, notably of the telencephalon (including cerebral cortex), gives five vesicles that produce the major anatomical and functional subdivisions of the brain and the ventricular system:

Vesicle	Structures	Ventricle
Telencephalon (endbrain)	Cerebral cortex, basal nuclei	Lateral ventricle
Diencephalon (interbrain)	Thalamus, hypothalamus, epithalamus	Third ventricle
Mesencephalon (midbrain)	Midbrain	Cerebral aqueduct
Metencephalon (afterbrain)	Pons, cerebellum	Fourth ventricle
Myelencephalon (spinal brain)	Medulla	Fourth ventricle

Flexures

Because the brain grows more rapidly than the skull, flexures develop to maximise the brain’s surface-to-volume ratio:

• Cephalic flexure pushes the mesencephalon (midbrain) upwards.
• Cervical flexure lies between brain stem and spinal cord.
• Pontine flexure generates the fourth ventricle.

By birth the cephalic flexure (at midbrain level) remains prominent. The staged pictures run from 5 weeks (vesicles plus midbrain and cervical flexures on the neural tube), to 13 weeks (folded brain with cerebral hemisphere, diencephalon, midbrain, cerebellum, pons, medulla oblongata, spinal cord), to birth.

Surface folding and the functional columns

• By 26 weeks the cerebral hemispheres grow rapidly and the surface starts to fold, producing convolutions. The progression of folding is shown at 25, 35, 40, 50 and 100 days, then 5, 6, 7, 8 and 9 months.
• In cross-section the developing neural tube is divided by the sulcus limitans into a dorsal alar plate and a ventral basal plate, with roof plate and fourth ventricle; the labelled functional columns from dorsal to ventral are somatic sensory, special visceral sensory (taste), general visceral sensory, general visceral motor (parasympathetic), special visceral motor (branchiomotor) and somatic motor.
• Brainstem and cranial nerves are covered in Reynolds’ lecture.
• The ventricles and basal nuclei “follow” the cerebral cortex: across 4, 7 and 9 months the corpus striatum and lateral ventricle reshape until at 9 months there are head and tail of caudate nucleus, lentiform nucleus, and frontal, temporal and occipital horns of the lateral ventricle.

Formation of the 6-layer cerebral cortex

The mature cerebral cortex has 6 layers (I-VI), running from the pia surface outermost to the ventricular surface. Within a layer all cells have similar properties:

• Same shape (for example pyramidal).
• Same axon trajectory (for example projection versus association).
• Same inputs (for example from thalamus).

Starting with a single neuroepithelium, the entire 6-layered adult cortex is produced by three processes:

1. Neurogenesis. Radial glial progenitor cells in the ventricular zone divide to produce newborn neurons.
2. Radial migration. Newborn neurons migrate outward from the ventricular zone, through the subventricular zone, along radial glia towards the pia.
3. Cortical organisation. Neurons settle in the cortical plate in birth-order-dependent bands, early-born, middle and late.

Malformations, one per step

Each of the three cortical malformations below maps onto failure of a different stage, which is the point of the sequence.

Disorder	Cellular cause	Genetics	Phenotype	Clinical consequences
Microcephaly (small brain)	Defective progenitor cell proliferation	Autosomal recessive mutation in one of 4 genes regulating cell division: ASPM, CDKRAP2, CENPJ, microcephalin	Reduced cell number, small brain size (greater than 2 SD below normal), morphologically normal	Developmental delay, epilepsy (rare)
Periventricular heterotopia	Defective early migration of newborn neurons	Mutation of filaminA (FLNA) or ARGEF2 (first identified by Prof Stephen Robertson, Otago)	Newborn neurons cannot migrate properly, forming nodules near the ventricle	Seizures in late adolescence, normal intelligence, dyslexia, behavioural problems (mood swings, anxiety, impulsiveness)
Subcortical band heterotopia (double cortex syndrome)	Defective later migration of newborn neurons	Mutation in doublecortin (DCX; X-linked), predominantly a disease of females	Newborn neurons accumulate in a band within white matter below the cortex (“double cortex”); shallow sulci, partial cortex develops	Severe epilepsy, profound developmental delay, intellectual disability, motor deficits

MRI appearances: microcephaly shows a visibly smaller brain than an age-matched normal with relatively enlarged CSF spaces; periventricular heterotopia shows grey-matter-signal nodules adjacent to the lateral ventricles; subcortical band heterotopia shows a band of grey-matter signal within the white matter beneath the cortex.

Formation of neural circuits

Axon growth and navigation

• Growth cone. A finger-like structure at the end of a growing axon that actively explores the environment to detect guidance cues.
• Guidance cues. Molecules in the extracellular matrix that either attract or repel the growth cone, dictating the axon’s direction.
• Signal transduction. When the growth cone encounters a guidance cue it triggers intracellular signalling pathways that change the cytoskeleton, causing the axon to extend or retract.
• A pioneer neuron navigates first past repulsive and attractive cues and past guiding cells; follower neurons fasciculate with it, and branches extend from the main branch to the target.

Congenital mirror movements

• A rare genetic disorder with involuntary mirroring of movements from one side of the body to the other.
• Cellular cause: failure of axon tracts to grow to the correct side.
• Genetic causes: mutations in DCC (receptor for an attractive cue) and Rad51 (unknown function).
• Phenotype: inability to move the right and left hands or fingers independently.
• Failed development of neural circuits is believed to underpin Autism Spectrum Disorder and other neurodevelopmental disorders.

Spinal cord and the peripheral nervous system

• The spinal cord forms from the caudal neural tube. In cross-section: dorsal root ganglion (sensory neurons, from neural crest), alar plate giving dorsal horn interneurons, basal plate giving ventral horn motor neurons, surrounding white matter, and the central cavity.
• The entire PNS forms from the neural crest; these cells migrate away from the dorsal neural tube and into the body, dorsally and ventrally.

Timing and path determine neural crest fate

Path	Timing	Derivatives
1. Medial	Early migration	Sympathetic neurons and glia; adrenal (medulla) chromaffin cells
2. Intermediate	Mid migration	Sensory neurons (DRG); glia (Schwann cells) of spinal nerves
3. Lateral	Late migration	Pigment cells (melanocytes)

Parasympathetic postganglionic neurons come from neural crest that migrates along parasympathetic preganglionic axons.

Abnormalities of neural crest development

• Hirschsprung disease (intestinal aganglionosis or megacolon). A rare birth defect in which nerves are missing from the large intestine, usually affecting the rectum or rectosigmoid region, due to failed migration of neural crest cells into specific gut segments. The intestine cannot move stool normally, leading to constipation and intestinal obstruction.
• Neuroblastoma. A common childhood cancer developing in nerve tissue, most often in the adrenal glands, beginning in infancy. It results from loss of control of proliferation of a subpopulation of neural crest cells.

After birth: maturation

• At birth the external shape of the brain and spinal cord is well developed. What follows is increased brain size, increased synapses and proper wiring.
• Myelination continues up to the early 20s, about 2 years earlier in females, and progresses over the cortical surface from posterior to anterior across ages 5, 8, 12, 16 and 20 years.
• The brain finishes developing and maturing in the mid-to-late 20s; the prefrontal cortex is one of the last parts to mature.

Adverse environments in the final stages

The prefrontal cortex, last to complete development, governs emotion, judgment, consequences of actions, motivation and personality. It can be affected by:

• Alcohol and drugs.
• Childhood or adolescent stress (neglect, abuse, exposure to inappropriate family or neighbourhood environments).

Impairment of brain development brings increased risk for neuropsychiatric disorders: developmental delay, depression, schizophrenia, and personality and emotional disorders.

Timeline of human brain development

Key milestones with their timings:

• Specification of neural ectoderm: about 14-16 days.
• Neural tube closure: anterior about 26 days, posterior about 28 days.
• Cerebral cortex expansion: about 35 days to birth.
• Neural circuit formation: about 40 days to the early years.

Across the whole span from conception through gestation (weeks 4 to 32+), birth, adolescence and adulthood, the overlapping processes are neurulation, neurogenesis, neuronal migration, apoptosis, synaptogenesis, gliogenesis, myelination (extending furthest into adulthood) and experience-dependent synaptic pruning (from mid-gestation into adolescence).

Prenatal origins of neuropsychiatric disease

Important

The human brain is vulnerable to injury during early neurodevelopment that begins in the intrauterine period but may not manifest until later in life. Intrauterine exposures at key developmental timepoints may increase an individual’s risk of neuropsychiatric disease.

Mechanisms of fetal brain injury or maldevelopment, as a chain:

• Maternal health and exposures: chronic medical conditions, maternal stress, mental health disorders, substance use, environmental or toxin exposure.
• Potential mechanisms: hypoxia, infection, inflammation.
• Neurodevelopmental consequences: brain injury, behavioural and cognitive impairments, neuropsychiatric disorders.

Maternal immune activation (MIA)

MIA is mediated by activation of inflammatory pathways, producing increased cytokines and chemokines that cross the placental and blood-brain barriers and alter fetal neural development. Its risk profile by life stage:

• Mother: immunological activation from infection, autoimmunity and genetic predisposition; increased IL-6, activation of Th17 cells, increased IL-17.
• Gestation: fetal immune status plus genetic composition helps determine vulnerability to MIA.
• Childhood: MIA offspring have heightened risk for autism spectrum disorder.
• Adolescence: MIA offspring are more susceptible to “second hits” induced by stress and drug abuse.
• Adulthood: MIA offspring have heightened risk of psychiatric and neurologic disorders.

By compartment: maternal (systemic immune activation, increased cytokines) -> placental (barrier compromised, allowing passage of maternally derived cytokines) -> fetal (activation of fetal immune responses, developmental disturbances, structural disturbances).

SARS-CoV-2 as a current example

• Serum proteomic profiling of mother-infant dyads with in utero SARS-CoV-2 (Cell Reports Medicine 2021) found that prenatal SARS-CoV-2 infection triggers NF-$\kappa$B-dependent immune activation, that pregnant women with severe COVID-19 show antiviral IFN-$\lambda$ signalling, that infection re-shapes maternal immunity at delivery, and that COVID-19-exposed infants show altered neonatal immunity at birth. The cohort sampled maternal blood at diagnosis (healthy n=18, COVID19+ n=79) and at delivery (healthy n=14, COVID19+ n=49), infant blood (healthy n=7, exposed n=45) and cord blood (healthy n=14, exposed n=32): 258 specimens, 1440 proteins. The authors stress long-term postpregnancy clinical follow-up of mother-infant dyads.
• The neurologic manifestations of SARS-CoV-2 (headache, dizziness, confusion, altered mental status, olfactory dysfunction), with reports of encephalitis and positive CSF samples, raise the question of direct viral neurotropism. Potential risk for schizophrenia later in life is supported by evidence from prior pandemics and from MIA models; proposed routes of increased risk are vertical transmission, exposure to inflammatory markers in utero, and timing of exposure. Long-term impact on offspring, and transgenerational effects, remain open questions.
• Comparing MIA models and clinical literature against emerging SARS-CoV-2 data: immune system stimulation (via RNA and TLRs), cytokine elevations (IL-6, IL-17) and perinatal complications are shared, while effects in offspring (behaviour, mood, cognition) remain unanswered for SARS-CoV-2.

Self-test

1. Describe the steps of neurulation from flat ectoderm to closed neural tube, with the approximate days.
2. List the four fates of the neural tube and its lining, and state what the neural crest gives rise to.
3. A neonate is born with the forebrain absent and brain tissue degenerated. Name the condition, explain which closure event failed and when that event should have occurred.
4. Distinguish spina bifida occulta, meningocoele and myelomeningocoele by what herniates and by clinical consequence.
5. What daily folate dose is advised in pregnancy or when trying to conceive, and why does folate matter for the developing neural tube?
6. Explain why the rostral end of the neural tube expands into vesicles rather than staying tubular.
7. List the five secondary brain vesicles with their derived structures and associated ventricle.
8. Name the three flexures, state what each achieves, and say which remains prominent at birth.
9. Describe the three processes that turn a single neuroepithelium into the 6-layered cortex.
10. Predict the malformation that results if progenitor proliferation fails, if early neuronal migration fails, and if later migration fails; name the causative gene(s) for each.
11. Distinguish periventricular heterotopia from subcortical band heterotopia by MRI appearance, inheritance and cognitive outcome.
12. Describe how a growth cone determines the direction an axon grows.
13. What happens to axon tracts in congenital mirror movements, which genes are implicated, and how does the patient present?
14. List the three neural crest migration paths with their timing and derivatives.
15. A newborn has failure to pass stool and intestinal obstruction; nerves are absent from the rectosigmoid region. Name the condition and its cellular cause.
16. Until what age do myelination and overall brain maturation continue, and which cortical region matures last?
17. List the factors that can derail the final stages of prefrontal cortex development, and the risks that follow.
18. State the four timeline milestones of human brain development with their timings.
19. Describe the maternal, placental and fetal compartment events in maternal immune activation, naming the cytokines involved.
20. Integrative: a mother has a severe infection at 4 weeks of gestation and another at 30 weeks. Using the developmental timeline, explain why the likely consequences differ.

Answers

Reveal answers

1. The ectoderm thickens along the midline axis to form the neural plate (about 19 days); the plate invaginates into a groove flanked by neural folds (about 20 days); the plate folds inward and neural crest appears at the fold margins (about 22 days); the folds fuse into the neural tube, separate from surface ectoderm (about 26 days).
2. Rostral tube forms the brain, caudal tube forms the spinal cord, the cavity forms the ventricular system, and the epithelial cells lining the tube generate all CNS neurons. The neural crest gives rise to neurons destined to reside in ganglia and to the entire PNS.
3. Anencephaly. The rostral neural tube failed to fuse, so the developing brain stayed in contact with amniotic fluid and degenerated; the anterior neuropore should close at about 24-26 days.
4. Occulta: no herniation, a bony defect only, asymptomatic, sometimes a dimple, hair patch or red mark at the base of the spine, about 10% of the population. Meningocoele: a sac of spinal fluid herniates but cord and nerves stay in place, minor disabilities. Myelomeningocoele: the spinal canal does not close and cord and nerves are external, with motor and sensory problems below the sac plus bowel and bladder problems.
5. 0.4-0.8 mg/day. Folate (vitamin B9) is required for DNA metabolism in rapidly dividing cells, and consumption during pregnancy is associated with reduced NTD rates.
6. Rostral cells divide faster than caudal cells and CSF production is increased anteriorly, so the rostral-most end expands.
7. Telencephalon: cerebral cortex and basal nuclei, lateral ventricle. Diencephalon: thalamus, hypothalamus, epithalamus, third ventricle. Mesencephalon: midbrain, cerebral aqueduct. Metencephalon: pons and cerebellum, fourth ventricle. Myelencephalon: medulla, fourth ventricle.
8. Cephalic flexure pushes the mesencephalon upwards; cervical flexure lies between brain stem and spinal cord; pontine flexure generates the fourth ventricle. They develop because the brain grows faster than the skull, maximising surface-to-volume ratio. The cephalic flexure at midbrain level remains prominent at birth.
9. Neurogenesis: radial glial progenitor cells in the ventricular zone produce newborn neurons. Radial migration: those neurons migrate outward along radial glia towards the pia. Cortical organisation: they settle in the cortical plate in birth-order-dependent layers, early, middle and late.
10. Failed progenitor proliferation gives microcephaly (ASPM, CDKRAP2, CENPJ, microcephalin). Failed early migration gives periventricular heterotopia (FLNA or ARGEF2). Failed later migration gives subcortical band heterotopia or double cortex syndrome (DCX).
11. Periventricular heterotopia: nodules of grey matter adjacent to the lateral ventricles, FLNA or ARGEF2, intelligence normal though with dyslexia, behavioural problems and seizures in late adolescence. Subcortical band heterotopia: a band of grey matter within white matter below the cortex with shallow sulci, X-linked DCX and predominantly female, with severe epilepsy, profound developmental delay and intellectual disability.
12. The growth cone, a finger-like structure at the axon tip, actively explores the environment for guidance cues; attractive or repulsive extracellular matrix molecules bind it and trigger intracellular signalling that changes the cytoskeleton, so the axon extends or retracts and thereby turns.
13. Axon tracts fail to grow to the correct side. Mutations in DCC (receptor for an attractive cue) and Rad51 (unknown function). The patient cannot move the right and left hands or fingers independently, with involuntary mirroring of movement from one side to the other.
14. Medial path, early: sympathetic neurons and glia, and adrenal medulla chromaffin cells. Intermediate path, mid: sensory neurons of the DRG and Schwann cells of spinal nerves. Lateral path, late: pigment cells (melanocytes).
15. Hirschsprung disease (intestinal aganglionosis or megacolon), caused by failed migration of neural crest cells into specific gut segments so the intestine cannot move stool normally.
16. Myelination continues to the early 20s, about 2 years earlier in females; the brain finishes developing and maturing in the mid-to-late 20s, and the prefrontal cortex is one of the last parts to mature.
17. Alcohol and drugs, and childhood or adolescent stress including neglect, abuse and exposure to inappropriate family or neighbourhood environments. Risks: developmental delay, depression, schizophrenia, and personality and emotional disorders.
18. Specification of neural ectoderm about 14-16 days; neural tube closure anterior about 26 days and posterior about 28 days; cerebral cortex expansion about 35 days to birth; neural circuit formation about 40 days to the early years.
19. Maternal: systemic immune activation with increased cytokines, increased IL-6, Th17 activation and increased IL-17. Placental: the barrier is compromised and allows passage of maternally derived cytokines. Fetal: fetal immune responses are activated, producing developmental and structural disturbances.
20. At 4 weeks the tube is closing (anterior about 26 days, posterior about 28 days) and neurulation and early neurogenesis are the active processes, so insult risks neural tube defects and gross malformation. At 30 weeks neurulation is long complete and synaptogenesis, gliogenesis, myelination and pruning dominate, so insult acts through maternal immune activation and inflammation on circuit formation, with consequences such as autism spectrum disorder risk in childhood and psychiatric or neurologic disorders later, which may not manifest until years afterwards.