Neural induction 1 Lecture 1

What is a zygote?

The fertilised egg that is the single cell from which all tissues will develop.

Explain Hox gene organisation across rhombomeres and what this gives rise to

· Hox genes from clusters Hoxa Hoxb and Hoxd expressed in particular pattern across rhombomeres -> each rhombomere unique in the combination of Hox genes it expresses

What are the axes of the neural tube?

A-P

D-V

The dorsal-ventral axis, which refers to the orientation of the neural tube, distinguishing between the back (dorsal) and belly (ventral) sides.

Describe the 3 stages of embryonic development

1. Repeat cell divisions of zygote -> blastula
2. More and more cells generated -> opening of cavity devoid of cells = blastocoel, hole forms = blastopore (connects blastocoel)
- there is a single cell layer which wraps all/most/some of embryo (future tissues)
3. Gastrulation: 3 main embryonic layers form: ectoderm, mesoderm, endoderm
- (animal cap of) Animal pole -> ectoderm, neurogenic region forms here
- Vegetal pole -> endoderm
- Cells that delaminate ectoderm and endoderm migrate through blastopore to embryo cavity -> mesoderm

Which region of the blastula gives rise to ectoderm?

animal cap which is an area around the animal pole of the blastula

Ectoderm will form either the epidermis or neural tissue

Describe an early experiment in Xenopus involving the animal cap

· Isolate animal cap before or after gastrulation, observe fate of cells in isolated animal cap
· Before gastrulation: isolated animal cap -> epidermis
· After gastrulation: isolated animal cap -> neural tissue
· Something has changed

What is the neurogenic region?

A region of the ectoderm near the blastopore from which the entire nervous system will develop (from neuroblasts)

What are the fates of the embryonic layers in vertebrates?

Ectoderm -> nervous system or skin
Mesoderm -> muscle and bone
Endoderm -> internal organs

What are the 3 main neural inducers/genes?

Noggin, chordin, follistatin

How is the neurogenic region formed?

· During gastrulation: as mesoderm organises into layer, folds under ectoderm in the involuting marginal zone...
· Mesoderm releases neural inducers - noggin, chordin, follistatin
· Nearby ectoderm acquires neural identity, forming neurogenic region

What did grafting experiments by Spemann and Mangold discover?

· Discovered that a small region of the mesoderm near blastopore held unique powers of induction

· Graft patch of tissue from pigmented donor embryo around blastopore on a region far blastopore from host embryo

· Induction of 2 body axes each with its own neural tube · Secondary body axis formed of pigmented (donor) and non-pigmented (host) cells: donor cells can organise host cells so that they form a new body axis

· Non-pigmented axis so induction

· Area termed the 'Spemann and Mangold organiser'

What does the organiser region do to the mesoderm?

activates mesoderm to release neural inducers

What is the equivalent of the Spemann and Mangold organiser in mammals and invertebrates

mammals
- primitive node
invertebrates
cells that form the boundaries between the dorsoventral and anteroposterior compartments of the wing disc

What types of inductions were observed in sliced animal caps using different inducer molecules

· Direct: animal cap acquired neural identity
· Indirect: mesoderm was induced then secondarily induced neural tissue

Explain how Xenopus embryos were treated so that inducer molecules can be tested

Pre-gastrulation embryo
· UV light = ventralized embryo that lacks neural structures
· Li+ = hyperdorsalized embryo, oversized neural structures at expense of reduced ventral structures
(opposite effect)
· mRNA from Li+ treated embryo to UV treated embryo= embryo develops normally (ventralisation rescued)
· Use to test inducer molecules

What gene/protein can rescue ventralisation in UV treated embryo? How was this discovered? What is its role?

· Noggin

· mRNA as cDNA isolated from organiser region around blastopore

· Cloned into library, each type of cDNA amplified and tested for neural induction properties

· dorsalises embryo; Can reproduce rescue of ventralisation in UV treated embryo with single cDNA, noggin gene sufficient

· Noggin protein expression/ when applied to isolated animal cap can directly induct ectoderm to neural fate

· But is not the only neural inducer

What does chordin do?

· extracellular protein with potent axis-forming activities, including ability to recruit neighbouring cells into secondary axis (ventral side)

What does follistatin do?

· Follistatin protein binds to activin, preventing it from binding activin receptor
· Activin receptor = TM protein responsible for signal transduction that activates additional genes affecting induction of epidermal cell fate
· Truncated activin receptor prevents epidermal cell fate and instead induces neural cell fate

How is the role of follistatin demonstrated?

· Isolated blastula animal caps
· Left alone -> epidermis
· Injected with mRNA for truncated activin receptor -> neural cell fate

What are dissociated animal caps?

Animal caps sliced out and cells separated by manipulating Ca2+ levels

What was demonstrated during experiments with dissociated animal caps (before gastrulation)?

Dissociated animal cap cells
- differentiate into neurones; hence more genes other than noggin, chordin and follistatin participate in neural cell fate induction, and not just BMP blocking

- If exposed to BMP4 protein, differentiate into epidermal cells. Like intact cap (BMP via cell to cell interactions)

What do neural inducers - noggin, follistatin and chordin - do?

Block BMP receptor

Describe the current model of molecular mechanism for neural induction

· In ECM, BMP proteins bind to BMP receptors

· Via phosphorylation of Smad intracellular protein, BMP inhibits Zic1 gene; promotes epidermal cell fate

· But mesoderm releases noggin, chordin or follistatin proteins

· They diffuse through ECM of animal cap ectoderm -> block BMP

· BMP cannot bind BMP receptor, Smad not phosphorylated, Zic1 gene transcribed

· Zic1 and FGF protein (in extracellular matrix) activate ERK protein via FGF receptor -> activates transcription of Zic3 gene

· Zic1 and Zic3 promote transcription of neural genes and induction of neural cell fate

Why is neural induction broadly similar between vertebrates and invertebrates?

Evolutionary ancient process

What are the similarities between neural induction in insects and animals

Homologous genes
Drosophila
· Dpp gene = homologue of BMP gene
· Sog (short gastrulation) gene = homologue of chordin
· Blastula = similar organised; its an external epithelium
- future dorsal side = dpp expression dominates
- Future ventral side = will get invaginated to give rise to mesoderm, sog dominates
- Regions near invaginated mesoderm sit between dpp and sog domains; will become neurogenic region

Define neuroectoderm

Region of ectoderm that acquires neural fate

Neuroectoderm vs neurogenic region

neuroectoderm in vertebrates: all its cells will develop into the nervous system

neurogenic region in invertebrates: only neuroblasts of this region will give rise to the nervous system

What is the dorso-ventral inversion

· Invertebrates - ectodermal region -> nervous system = ventral
· Vertebrates = dorsal
· Dorso-ventral inversion

What are the differences between neural induction in invertebrates vs vertebrates

After gastrulation...

invertebrates e.g. Drosophila
· Nervous system seeded by neuroblasts = cells that delaminate from neurogenic region
· Neuroblasts (of the neurogenic region) detach from epithelium
· Start stereotyped sequence of asymmetric cell division, gives rise specific complements of neuronal progenitors
· These differentiate into all neuronal cells types and form nervous system

Vertebrates e.g. Xenopus, mammals,
· instead of invagination, neuroectoderm invaginates -> forms neural tube; will go onto form CNS
· Neural crest cells (a few cells from dorsal neural tube) do delaminate, will go onto form PNS
· Notochord releases retinoic acid and Shh -> formation of ventral plate in ventral side of neural tube

Define notochord

Mesodermal structure that runs the ventral length of the neural tube

releases molecules that modulate neuronal cell fate in the neural tube

What is different about neural induction during embryonic development about mammals than non-mammalian vertebrates (Xenopus/amphibian)

· Blastula and gastrula stages different
In mammals...
· Blastula has primitive streak instead of blastopore
· through primitive streak, migrating epiblasts enter blastocoel and are induced as endoderm
· Second wave of migration through primitive streak forms the mesoderm internally
· Once 3 embryonic layers formed, proceeding events similar

What are the general events that take place following blastula and gastrula formation in vertebrates (mammals and amphibians)

· Invaginating neural tube over mesodermal notochord
· Delaminating neural crest cells at interface between fully invaginated neural tube and dorsal epidermis

Define epiblasts

Undifferentiated cells on blastula surface

What is the dorso-ventral inversion

· Invertebrates - ectodermal region -> nervous system = ventral
· Vertebrates = dorsal
· Dorso-ventral inversion

What is the fate of the neuroectoderm cells?

· Anterior half -> brain and related structures
· Posterior half -> spinal cord (vertebrates), nerve cord (insects, invertebrates) and motor neurons to coordinate muscle groups in locomotion

How does the central nervous system form? What can defects in this process give rise to?

- Neurulation: Neural plate (thickened area of ectoderm) rolls up to form the neural tube
- Birth defects like anencephaly

How does the peripheral nervous system form?

From neural crest

What neural structures are formed in the Drosophila and where from?

· Anterior neuroblasts -> brain; subregions = protocerebrum, deutocerebrum, tritocerebrum
· Posterior neuroblasts -> oesophageal ganglion (like brainstem), thoracic and abdominal segments of nerve cord

Explain CNS development in mouse; what neural structures are formed and where from?

· Neural plate of the (neuro)ectoderm invaginates, forms neural tube along notochord
· Most anterior region of neural tube -> brain (prosencephalon, mesencephalon, metencephalon)
· Posterior of neural tube -> spinal cord

What are neural progenitors?

· cells that are capable of dividing a limited number of times and have the capacity to differentiate into a restricted repertoire of neuronal and glial cell types
· Neuroblasts in invertebrates

Explain distribution of maternal mRNA in the zygote cytoplasm, and what happens to it

· Heterogenous - differential distribution of maternal mRNA
· Translated into TFs that regulate transcription of further TFs -> TF cascade
· There are gradients of concentration of maternal mRNA along A-P axis of early embryo, diff TFs transcribed when enough activating TF protein
· -> French flag model

What Is the French flag model?

· Continuous gradient binarized from anterior to posterior

· Boundaries defined by concentration thresholds for activation of TF gene promotor / gene expression Ie high affinity to promotor = low conc of TF needed

What determines threshold?

· The specific concentration of transcription factors (TFs) required to activate gene expression, influenced by spatial distribution in the embryo.

What is genetic pathway for neural development across the A-P axis in invertebrates?

A series of gene regulatory events that involve maternal mRNA, transcription factors, and signaling pathways, coordinating the development of neural tissues along the anterior-posterior axis of invertebrates.

What is the result of this genetic pathway?

Combinatorial code emerges -> each body segment gets unique positional identity

What is combinatorial code?

Code of TF; differential/amount of gene expression in a cell
Upstream effect - TF environment -> diff TFs

What are gap genes, what is their role?

TFs whose expression pattern defines large chunks of the A-P axis of the embryo
Control activation of pair-rule genes

What experiment demonstrates the role of gap genes?

Gap gene knockout removes substantial/continuous portion of body regions

How are pair-rule genes expressed, what do they do?

In alternating pairs of body segments
Alongside other TFs, control segment polarity and homeotic genes

How are segment polarity genes expressed?

Narrow bands of cells of every body segment

What are homeotic genes, what is the most important one?

· Genes that drive homeosis (transformation of one organ to another)
· Hox genes

What is the role of Hox genes; which species have Hox genes?

· Pattern the A-P body axis of neural tube
· In all free-living animals

What is significant about Hox gene order in genome?

· Sequential order in genome corresponds with regional expression in the A-P axis: 3'->5' = anterior -> posterior

What roles do Hox genes play in different animals and why; how can this be demonstrated experimentally?

· Evolutionary ancient/conserved
· Similar/identical role in development
· DNA sequence for specific Hox gene in mammal can rescue function of homologous gene in Drosophila when knocked into its place

How many Hox gene clusters in vertebrate genome and why?

· 4 - abcd
· Vertebrate genome experience 2 rounds of whole-genome duplication during evolution of chordates

How many Hox gene clusters are there in Drosophila and why

· 4 - abcd
· Vertebrate genome experience 2 rounds of whole-genome duplication during evolution of chordates

How many Hox gene clusters are there in Drosophila and why

· 2
· Single Hox gene cluster split into 2 throughout evolution

What are the subdivisions of the mammalian hindbrain? How many? What emerges from them

8 rhombomeres, cranial nerves emerge from one/two adj rhombomeres

Explain Hox gene organisation across rhombomeres and what this gives rise to

· Hox genes from clusters Hoxa Hoxb and Hoxd expressed in particular pattern across rhombomeres -> each rhombomere unique in the combination of Hox genes it expresses

What is the effect of Hoxa knockout

· Hoxa expressed in r4
· Knockout causes loss of r4 and r5 -> abducens cranial nerve from r5 lost entirely and facial nerve (r4+r5) affected
· Cranial nerves emerging from other intact rhombomeres are still in tact

What is revealed by Hox gene knockouts?

Single gene can control positional identity and peculiarities of an organ or body region

What do Hox genes do to animals axis wise? How can this be demonstrated experimentally?

·Caudalize/posteriorize animal
· Knockout of whole Hox gene cluster, posterior segments resemble the most anterior segment of the body/regions acquire characteristics of first rhombomere

What are the 3 main stages of the development of the nervous system?

1. First signal activates/neuralizes gastrula ectoderm -> neuroectoderm
2. 2nd signal stabilises neural fate of a region in neuroectoderm, defines an anterior and posterior
3. 3rd signal transforms region of neural tube -> subregions = midbrain, hindbrain, spinal cord aka Hox gene caudalizes a region of the neural tube, detailing the subregions: midbrain, hindbrain, spinal cord.

What does retinoic acid (RA) do?

· Its gradient controls Hox gene expression along A-P axis
· High levels promotes posterior identity, low levels permit Hox genes for anterior identity

Where is RA released from/how?

· Notochord
· Releases little/non at anterior end, increasingly more in gradient towards posterior end

What is the mechanism for RA action

· Diffuses into developing neural tube
· Crosses cytoplasmic membranes
· Binds intracellular receptor
· RA receptor -> nucleus, binds RARE
· Hox gene activated

Different Hox gene (promotors) activated at different...

· Concentrations of RA
· Each region along neural tube, one or more Hox genes

What further narrows the A-P Hox gene expression along with RA

Combinatorial code narrows down identity of each neural tube region in the A-P axis

What gradients are there along the A-P axis?

· RA
· FGF

What is FGF and what does it do?

· Secreted by lateral mesoderm
· Regulate Hox gene expression in dose-dependent manner (inc from A->P)
· Refines positional identity of A-P regions of neural tube, differentially drives gene expression

What are the axes of the neural tube?

A-P
D-V

What controls D-V axis?

· Protein gradients like A-P
· Shh, BMP (reuse signal), Wnt

Explain how a unique D-V positional identity of neurones is established in the neural tube

1. Neural tube invaginates, notochord expresses Shh
2. Shh induces floorplate formation (most ventral region)
3. Floorplate releases Shh from ventral upwards
4. In parallel, neural tubule closes, epidermal Bmp induces dorsal part -> roof plate
5. Roof plate secretes BMP and Wnt from dorsal down
6. Opposing gradients of Bmp and Shh confer unique positional identity of neurones along D-V axis

What interaction at neural tube induces further differentiation?

· Interaction between dorsal part and epidermis mediated by BMPs
· Induces differentiation + migration of neural crest cells
· -> many cell types

Where do parasympathetic and sympathetic ganglia arise from?

· Neural crest cells
· Sympathetic = from NCC of trunk
· Parasympathetic = from NCC of head

How can the role of the notochord be determined experimentally?

· Removal of notochord prevents floorplate formation
· Transplanted notochord -> floorplate induction
· Placing ectopic notochord on lateral side induces ectopic floorplate [ectopic?]

What is the default identity of intermediate cells of the neural tube? How can this be demonstrated and manipulated experimentally?

· Extract portion of intermediate region
· Alone, cells adopt dorsal identity and express dorsal marker genes e.g. Pax7
· Add Shh, cells adopt ventral identity; express ventral marker genes e.g. Olig2, Nkx2.2

Compare genes patterning the D-V axis in mouse and fly

· Evolutionary ancient so similar
· Msh, Ind, Vnd in fly = homologues of Msx, Gsh1 and Nkx2.2 in mouse
· In Drosophila embryo, D-V pattern in neuroectoderm; in mouse embryo, same D-V pattern in neural tube (Dorsal to ventral)

What is the most dorsal part of the embryonic neuroectoderm?

Bottom of neural tube because neural tube invaginated

How do invertebrates and vertebrates compare dorso-ventrally and why

· Dorso-ventrally inverted
· But invagination of neural tube reverses relative position of the regions expressing the genes, matching with the absolute positions of their homologues in the fly