Lecture 5- Neurulation: Mechanisms of neural tube formation Flashcards
What is neural induction?
-the specification of the dorsal ectoderm to a neural fate
What is neurulation?
-embryonic process that generates the neural tube
What does the neural plate undergo during development into the neural tube?
- morphogenic changes: shaping, bending and folding
- neural plate bends and edges elevate
- neural crest migration is the final step in the neural tube closure
Where are the presumptive/future neural crest, and epidermis in relation to the neural plate?
-the neural folds fuse in the midline
How does amphibian neuralation differ from human neuralation?
-in amphibians neural tube closes as a simultaneous event along all neuroaxial levels
What does neural plate do during neuralation?
-bends and elevates
What do the dorsal margins of the neural tube do at the end of neuralation?
-fuse together “zip”
Is the mechanism of neural tube formation similar in humans and mice?
- yes
- the timing is different but the principle is the same
What is a neuropore?
- marks where neuralation is occuring
- openings in the closing neural tube
- reduce in size as neural tube closure proceeds
What are the three neuropores in a mouse embryo?
- Hindbrain neuropore
- Rostral/anterior neuropore
- Posterior neuropore
Where is neuralation initiated?
-at multiple sites
What is a closure site?
-position where neuralation is initiated
What signifies the end of neuralation?
-neuropore closure
How many closure sites are there in a mouse embryo?
-3
Where is neuralation initiated? (mouse embryo)
-at closure 1 in both directions
How many neuropores and closure sites are there in a human embryo?
- 2 neuropores, anterior and posterior
- 2 closure sites (1 and 3)
- existence of closure 2 in humans is controversial
What does failure of neural tube closure cause?
-neural tube defects NTDs
What are the three most common neural tube defects in humans and why do they arise?
- Craniorachischisis: failure of initiation from closure 1, completely open from midbrain to spine, lethal
- Anencephaly: failure of rostral (anterior) neuropore closure, lethal
- Spina bifida: failure of caudal (posterior) neuropore closure, wheelchair-bound, loss of function below the lesion
What is anencephaly a result of (mouse)?
-failure of initiation from closure 2 or failure of anterior neuropore closure
What is open spina bifida (myelomeningocele) in a mouse a result of?
-failure of posterior neuropore closure
What is craniorachischisis a result of? (mouse)
-failure of initiation from Closure 1
What happens to the exposed neural tissue when the neural tube fails to close?
- results in degeneration of exposed neural tissue in utero
- this is why in spina bifida you have a loss of function
- with anecephaly, the brain develops but degenerates in utero
How common are neural tube defects in humans and what are the two most common defects?
- 1 in every 1000 pregnancies
- spina bifida and anencephaly
What is the cause of neural tube defects?
- multifactorial and complex
- major genetic component in NTD susceptibility (but pattern of inheritance is sporadic)
- many candidate genes
- most human cases predict to involve multiple genes and a contribution from environmental factors
What is used as the primary prevention of neural tube defects in humans?
-maternal intake of folic acid
Can neural tube defects be repaired surgically?
- possible for early open spina bifida in utero
- carries many risks
- rate of miscarriage is high
What happens to the neural tube during neuralation?
-neural plate is shaped, bent, and fused along the midline
What are the four cellular and molecular steps of neuralation?
- neural plate shaping: cellular- convergent extension, molecular- planar cell polarity pathway (PCP)
- Elevation and apposition: cellular- hinge point formation, molecular- sonic hedgehog BMP pathway
- Adhesion and fusion: cellular- dorsal midline fusion of neural folds, cell protrusion, molecular- adhesion molecules
- Closure and remodelling: cellular-separation of the neural tube and surface ectoderm
What happens during 1. Neural plate shaping? (cellular)
- neural plate becomes narrower and longer, broad cranial region, narrow spinal region
- convergent extension: narrowing and lengthening of tissue without additional cell growth
- tissue undergoes simultaneous narrowing (convergence) along one axis and lengthening (extension) along another axis
What are the processes in 1. Neural plate shaping? (2)
- Collective migration: cells move in one direction as a cohesive sheet, no neighbour exchange, neural plate cells move towards the midline of the embryo
- Cell intercalation: cells change shape and exchange neighbours, neural plate cells redistribute in the anterior-posterior axis
What process does cell intercalation involve?
- protrusion of cellular processes
- cells form polarised protrusions (lamellpodia)
- protrusions attach and crawl on negbouring cells creating traction and tension
- tension elongates and pulls cells between one another (intercalation)= narrower and longer tissue
How is convergent extension regulated at the molecular level?
- by the PCP (Planar Cell Polarity) pathway
- also called non-canonical Wnt signalling pathway
What is the PCP (or the non-canonical Wnt signalling) pathway like?
- the PCP pathway is activated via binding of Wnt to Fz (the frizzled receptor) on the cell membrane
- the Fz receptor recruits Dsh (Dishevelled protein) which uses its PDZ and DEP domain to form a complex with Dishevelled-associated activator of morphogenesis 1 (DAAM1)
- DAAM1 then activates the protein Rho
- Rho activates Rho-associated kinase (ROCK) that is one of the major regulators of the cytoskeleton
- Dsh also forms a complex with rac1 and mediates profilin binding to actin
- rac1 activates JNK and also leads to actin polymerisation
- this all leads to changes in planar cell polarity
Where was PCP pathway first defined?
- in Drosophila
- the hairs are direct representation of neurons, the PCP pathway affects their polarity
- PCP pathway regulates the prganised polarity of epidermal cells in the Drosophila wing
- when you create a dsh mutant, the wing hair is non-organised
What gives cells polarity?
-restricted distribution of PCP signalling
What happens to cell polarity if you misregulate Dsh?
-wrong shape of cells
What is the connection between convergent extension and neural tube closure?
- convergent extension is required for initiation of neural tube closure
- mouse mutants fail to initiate closure from Closure 1 and develop craniorachischisis
- mutant genes encode proteins in PCP pathway, so get disruption of PCP signalling pathway
- no convergent extension of the neural tube
- neural tube is too broad to achieve closure
What is the medial hinge point (MHP) in the 2. Elevation and apposition processes?
-overlies the notochors; bending of the upper neural plate
What are the dorsolateral hinge points in 2. Elevation and apposition processes?
-point of attachment of the surface ectoderm and each neural fold; bending of the intermediate and lower neural plate
What does the bending of the neuroepithelium in 2. Elevation and apposition involve?
- changes in cell shape
- neuroepithelial cells transform from spindle shaped to wedge shaped
- apical narrowing and basal expansion
- cell wedging is essential for neural plate bending
What is the cellular mechanism for neural plate bending: Hypothesis 1?
- Apical organisation of cytoskeletal actin-myosin microfilaments
- purse string contraction of acto-myosin, reduction in the apical surface area
- tested by disrupting the cytoskeleton by actin-disassembling drugs, this resulted in cranial NTDs
- role in spinal neuralation remains unclear as the subjects treated woth actin-disassembling drugs did not develop spina bifida
What is the cellular mechanism for neural plate bending: Hypothesis 2?
- cells are widest at the position of the nucleus
- cell cycle dependent variation in the apico-basal position of the cell nuclei
- requirement for cell cycle regulation and nuclear migration remains unclear, so far untested
What regulates the bending of the neural plate? (2. Apposition and elevation)
- extracellular signalling
- BMP signalling inhibits DLHP formation
- sonic hedgehog, an intracellular signalling pathway, inhibits DLHP in the upper spine
How does 3. Adhesion and fusion work?
- few NTD models of adhesion/fusion
- molecular mediators poorly understood
- cell protrusions extend from the apical tips of the neural folds and provide cell-cell recognition and initial cell adhesion
- neural fold midline fusion is thought to be achieved by adhesion molecules
How does 4. Remodelling work?
- separation of the neural tube and surface ectoderm is caused by differential adhesion of the two tissues
- neural tube expresses neural cell adhesion molecule and neural (N)-cadherin
- surface ectoderm expresses epithelial (E)-cadherin
- cells will stick to the cells that have the same adhesion molecule. here have different so won’t stick together
- disruption of N-cadherin expression in Xenopus causes defective neural tube closure
- N-cadherin not essential for mammalian neuralation
What is primary neuralation?
- neural tube formed by shaping, bending and midline fusion
- forms brain and majority of spinal cord
- failure results in open neural tube defects
What is secondary neurulation?
- neural tube formed by cellular condensation and canalisation
- forms the lowest part of the spinal cord
- failure results in closed neural tube effects
What does the failure of secondary neurulation result in?
- closed neural tube defects
- spinal dysraphis: closed NTD
- least severe and least well defined group of NTDs
- spinal cord is tethered to other tissues
- faulty tissue separation
What is the cellular mechanism of secondary neurulation?
- mesenchymal cells condense and differentiate into neuroepithelium, generate the central lumen in the lower part of the neural tube
- cells reorganise “canalisation” to form the central lumen of the secondary neural tube
- lumen is continuous with primary neural tube
What is the primary prevention of human NTDs?
-folic acid intake during the peri-conceptional period (women)
What are the curent recommendations for folic acid intake?
- 400 microgrammes per day for all women during the peri-conceptional period
- folic acid 4-5 mg per day for all women at high risk of NTD
- it is ensured by mandatory folic acid fortification in food
Are many mothers folate deficient?
-no
Do we understand how folic acid prevents NTDs?
- very limited at best
- folates are integral to one-carbon metabolism which produces pyramidines and purines for DNA synthesis
- exogenous folic acid stimulates a cellular response to enable developing embryo to overcome NTD inducing genetic or environmental effects
Are all human NTDs prevented by intake of folic acid?
- no
- a subset of NTDs are not prevented by folic acid supplementation
What is the PONTI trial about?
- testing if Inositol is successful in preventing NTDs
- Inositol is the only vitamin-like molecula required for normal neural tube closure in rodents
- some human NTD pregnancies have lowe maternal inositol concentrations than unaffected pregnancies
What is the commonly used mouse model used for human neural tube defects?
- Curly tail mouse
- spontaneous mutant, partially penetrant NTDs
- 15-20% spina bifida, 5% exencephaly
- pathogenesis resembles human NTDs
- non-responsive to folate acid
- penetrance strongly influenced by genetic background and environmental factors
What is the mechanism of spina bifida in the Curly tail mouse?
- reduce cell proliferation in hindgut
- increased curvature of body axis due to different rates of growth, mechanically delays the closure
- delayed closure of neural folds
- spina bifida and tail defects
treatments: -rebalancing growth corrects spina bifida - splinting normalize neural tube closure
- inositol prevents spina bifida
Grhl3 is the main causative gene
What is Grainyhead-like genes needed for?
- correct levels of Grainyhead-like gene 2 and 3 expression are required for normal neuralation (mice)
- now looking for the Grainyhead-like 2 and 3 regulatory mutations in humans
What is the causative genetic defect in the axial defects mouse?
- up-regulation of Grhl2
- reduction in Grhl2 expression rescues Axial defect phenotype
additional information:
- adhesion proteins are miss-regulated in axial defect mouse model too
- another adhesion
the surface ectoderm is too adhesive ti allow correct adhesion fusion and or remodelling
-GRHL2 may regulate adhesion proteins
