M&P1988,2-21

2-Zell Stadium einer Zygote in vitro

Brain development: An introduction

A

Brain development: An introduction

rosemarie.grantyn@charite.dewww.charite.de/ch/physio/sep/research-rg

(with password 'rgmns0405')

A Dec04 - Induction of neuroectoderm & earlyneural morphogenesis- Regional specification & factors that determineidentity of individual neurons and glial cells

B March05 - Axon outgrowth, target innervation, synaptogenesis- Regressive events, competitive interactions, lesion and repair in the CNS

Recommended reading:

1) Nicholls JG, Martin AR, Wallace BG, Fuchs PA (2001) From Neuron to Brain, Sinauer, Sunderland, MA (580 pp.)

2) Sanes DH, Reh TA, Harris WA (2000) Development of the Nervous System, Academic press, San Diego (500 pp.)

3) Christ B, Wachtler F (1998) Medizinische Embryologie, UllsteinMedical, Wiesbaden (348 pp.) (German)

4) Moore, KL, Persaud TVN (1998) The Developing Human, Saunders, Philadelphia (563 pp.)

5) Gilbert SF (2003) Developmental Biology, Sinauer, Sunderland,MA (838 pp.)

6) Squire LR, Bloom FE, McConell SK, Roberts JL, Spitzer NC, Zigmond MJ (2003) Fundamental Neuroscience, Academic Press,Amsterdam, Boston etc. (1426 pp.)

Q1: Why and when do developmental abnormalities arise?

Frequency and causes of human congenital anomalies

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3% of newborns - significant anomalies (such as meromelia, microcephaly)

14% of newborns - smaller single defects(such as color blindness, abnormal pigmentation, lighterforms of mental retardation etc. )

About 25% of anomalies are due to a combination of genetic and environmental factors

50-60% of all cases of spontaneous abortion -chromosomal anomalies

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3th-5th week: Highest risk during the period of organogenesis

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Example: Severe anomalyof limb developmentafter the influence ofthalidomide (= Contergan)

Amelia

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MeromeliaM&P98

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Example: Additional fingers/toes

C, D - polydaktyly, E, F - partial duplication of foot or numb

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Meroanencephaly, spina bifida

A bit of terminology...

Embryonic period: 1st-8th weekzygote

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blastocyst

gastrula (w 3 germ layers)

At the end of the embryonic period the heart and circulation are functioning

Fetal period: 9th week till birth

Prenatal, postnatal periodDevelopment is not over at birth: the brain triples its weight between birth and 16 years of age

1. Why and when do developmental abnormalities arise?

1st summary:

- About 15% of the developmental anomalies are due to gene defects.Another 10% are due to environmental factors (viral infections, alcohol, poisons), the remaining 75% are of multifactorial or unclear origin.

- The development of a new human being comprises the embryonic period(1st till 8th week), the fetal period (9th till 28 week) and the postnatalperiod. During the 4-5th period of gestation the risk of a developmental anomaly is highest.

- Both the lack as well as the excessive or premature influence of a developmental signal can cause a developmental anomaly.

Q2: How is the CNS 'anlage' formed?

Does the genome encode all the properties of the organism/brain to be built?

M&P1988,2-21

2-Zell Stadium einer Zygote in vitro

The entire information for the development of amulticellular orgnizm is contained in the fertilized egg...

No, we have a so called generative, not a descriptive program of development. This means: Each of the actually active genes only generates the signals for the following stage of development.

Note: 1010-1012 cells in the brain, but only about 105 genes

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1) Cell division (w or wo cell growth)

2) Pattern formation (evolvement of the body building plan, establishment of rostro-caudal and dorso-ventral axes, limb axes)

3) Cell differentiation (as a consequence of induction)

4) Growth & changes in body/organ shape

5) Activity-dependent adjustment of structure and function, compensation of small developmental anomalies

The development of a multicellular organism comprises the following processes

that may occur in parallel

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1st week:Formation of the blastocyst

The daughter cells (blastomeres) become smaller with each division, 1 cycle requires about 20-24 h. The generation of new mRNA (due to translation) still occurs under the control of maternal genes ('maternal factors').

A completely new offspring is present when the translation is completely under the control of the embryonic genome (end of first week?) >ethical considerations

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Primitive node(Hensen's node)Primitive streak

Embryonic disc

Connecting stalk

The equivalent to Hensen's node (in chick and mammalian embryos)is the Spemann organizer (in amphibians)

Transfer of Hensen's node from a quail embryoto a chicken embryo

Induction of an additional body axis in the host

embryo

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Polarity: Differential morphological properties along an axis Induction: A small group of cells determines the properties of the neighboring tissuePrimary induction: Induction of the entire body axis by a group of cells(organizer cells)

Nobel price for medicine 1935 to Spemann

The concept of the organizer was introduced by Hilde Mangold und Hans Spemann on the

basis of transplantation studies in amphibians

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The genes of the organizer encode secreted proteins, receptor protein kinases and transcription factors that drive the further fate of the neighboring tissue

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Primitive streak

Embryonic disc

Connecting stalk

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Transcription factors bind to the regulatory regions of one or more genes and influence

the rate at which these genes are transcribed

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What happens during the primary induction?

Sequential and hierarchical activation of genes encoding secreted factors and transcription factors

Master Gene: sonic hedgehog (e.g. shh)

Transcription factors (e.g. homeobox-containing genes, HOX)

Growth factors (e.g. FGF)

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The development of the body axis is a remarkable example of the homology between genes of invertebrates (drosophila) and vertebrates (amphibians, birds, mammals)

Endovaginal sonogramm ca. 3 weeks after conception:Now starts a particularly critical period of the embryonic development,

when defects in organogenesis may produce profound anomalies A - Amnionhöhle, YS - Dottersack, E - Endometrium

Aus: M&P1998

End of third week: At this stage we have a neural tube, with a rostral and a caudalneuropore, and a (still incomplete) number of somites

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Somites are compact aggregates of mesenchymal cells from which cells migrate to give rise to the vertebrae, ribs and the axial muscles

Size: 3 mm

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The precursor cells for the CNS (neural tube) and the PNS (neural crest) are now separate from each other

A 21 day old human embryo has 3 germ layers

Part of the ectoderm gives rise to the nervous system >neuroectoderm

Mesenchym (mesoblast): a loose network of embryonic connective tissue. Some mesenchym forms a layer called mesoderm. The mesenchym may influence the fate of cells emerging from the ectoderm.

2. How is the CNS 'anlage' formed?

2nd summary:

- The human development follows a generative developmental program.- A new offspring is present when the translation is completely under the control of the embryonic genome.- In mammals the cells of Hensen's node contain genes that are sufficient to produce a complete body building plan. This area corresponds to Spemann's (body) organizer.- A group of hierarchically organized genes controls the formation of the body axis prducing secreted molecules (nodal, shh) or transcriptionfactors (Lim-1). The master genes are at the top of the hierarchy andinfluence many other genes.- In humans the body axis is defined at the end of the 3rd week ofgestation (with the rostral and caudal neuropores present, the somites and three germ layers. - The ectoderm has organized into the neural tube and the neural crest.

Q3: How is the regional specification of neural tissue achieved?

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tEnd of the 4th week:The rostral and caudal neuropores are fused,the full set of somites is present, the upper limb bud emerges.Otic pit (primorium of the

inner ear)

The embryo has a length of 4.5 mm

The brain gets organized into several vesicles thatreflect its increasing regional differences

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Midbrain-hindbrain organizer (WNT1, engrailed-1, FGF-8)

Hindbrain

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The notochord is a rod of cells of mesenchymal originthat will drive the differention of the neural tube cells.

In contrast to the rest of the vertebrate brain, the embryonic hindbrain (rhombencephalon)

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Several genes have been identified whose pattern of expression correlates withthe segmentary boundaries of the developing hindbrain

The 'activator-transformer hypothesis': Each segment (rhombomere) exhibits the same general pattern of neuron differentiation (default), but then in each segment the pattern is modified in specific ways.

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tionThe identity of each rhombomere is determined by the

differential expression of HOX genes

Homeotic genes are master genes that encode the expression of many other genes

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tyThe positional identity along the body axis is also based on a HOX code

How is a cranio-caudal positional signal generated ?

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Tissue

Gene IGene IIGene IIIGene IV

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A gradient of retinoic acidcontrols the differential rostro-caudal pattern of

HOX gene expression

(retinoic acid is also produced by Hensen's node, the Spemannorganizer)

Retinoic acid activatesthe transcription of HOX genes,

and different HOX genes have different sensitivity for retinoic acid

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The characteristics of the vertebrate nervous system vary not only along cranio-caudal axis but also along the dorso-ventral axis

Notochord

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In the spinal cord, a band of specialized glial cells, called the floor plate, drives the further development of the neurons in the ventral area (e.g. motoneurons);

the floor plate is, in its turn, induced by the notochordN

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If the notochord is missing, floor plate cells and motoneurons fail to form

shh always specifies the ventral phenotype, but the latter is differentin dependence on what HOX genes are turned on (from caudal to rostral: spinal motoneurons,dopaminergic neurons in the posterior hindbrain, serotoninergic neurons in rostral hindbrain, ocular motoneurons in the midbrain)

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As a rule of thumb, the fate of pluripotent precursor cells is first determined along the rostro-caudal axis

and then along the dorso-ventral axis

3. How is the regional specification of the neural tissue achieved?

3rd summary:- By the end of the 4th week the rostral and caudal neuropores areclosed. The regional differentiation of the brain vesicles and caudal neural tube proceeds.- In the brain the now operant organizer is the midbrain-hindbrainorganizer (transplantation leads to ectopic cerebellum and tectum; WNT1, en-1, FGF-8).-The embryonic hindbrain has a segmentl structure (7 rhombomeres).Each rhombomere exhibits a characteristic pattern of neurondifferentiation (default pattern), but then it is modified in a specific way uner the influence of particular, segment-specificgene expression patterns.- The identity of each rhombomere is determined by the differentialrostrocaudal pattern of HOX gene expression. The expression of the HOX genes is controlled by retinoic acid (higher conc. caudally).- The dorso-ventral polarity is controlled by shh (the notochordinduces floor plate cells, the floor plate cells induce the basal plate cells, like motoneurons).

Q3: How do single cells of the CNS acquire their phenotype?

The cells of the embryo/fetus are subdivided according to their developmental stage into

- stem cells (ominipotent, pluripotent)

- precursor cells (= already determined but not yet differentiated X-blasts)

- differentiated cells (= postmitotic cells, with clear phenotype)

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Lab case 1Embryonic stem cells also divide and differentiate under in vitro. They can be transpanted back into the maternal uterus .The supernumerary zygotes are a potential source for gaining defined cells for transplantation (blood, skin, neurons?) Em

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By injecting embryonic stem cells with a targetted mutation into a blastocyst one can generate transgenic animals with defined properties .Cells made to express a fluorescent protein under a specific promoter can be traced towards their final destination.

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During cell division the cell is near the ventricular surface, and the pial connection is temporarily lost. DNA synthesis occurs while the nucleus is near the pial cell surface

Following cell division, one or both of thedaughter cells may migrate away. This is the point when they become neurons or glial cells.

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Cells divide near the ventricular surface and then migrate away

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Once neurons migrate away from the ventricular zone, they becomepostmitotic, i.e. they will never divide again. In contrast, glial cell precursors can even divide after they have reached their final destination

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In cortical structures radial glial cells serve as a scaffolf for migration.Isoforms for the integrin family of receptors mediate migration.

Cells that later produce the GnRH migrate > 2 mm, from the olfactory placode into the thalamus

4. How do single cells of the CNS acquire their phenotype?

4th summary:

- Depending on the degree of determination, immature cells are subdivided into stemm cells (omnipotent, pluripotent), (partially) determined precursor cells, differentiated cells.- Cells divide near the ventricular surface and then migrate away.- The neuroepithelium (neuroectoderm) gives rise to neuroblasts >neurons, glioblasts > astrocytes, oligodendrocytes. Microglial cells are derived from the mesenchym.- Once a neuron migrates away from the ventricular surface it will never divide again. In contrast, glial cell precursors can even divide after they have reached their final destination.