1
Q
What is neurulation?
A
- Folding of neural plate which bends up and later fuses to form a neural tube that will differentiate into brain and spinal cord
- Occurs at 4th week of human gestation
- The CNS is derived from neuroectoderm
2
Q
What does the neural tube give rise to?
A
- Brain
- Spinal cord
- Cranial and spinal nerves
- Eyes and other sensory organs
- Neural crest
-Neural tube arises from ectoderm
3
Q
What are the steps of neurulation?
A
- Specification of neuroectoderm
- Neural plate formation
- Neural plate folding
- Neural tube closure
- Neural crest closure
- Neural crest migration
4
Q
What is the establishment of the anteroposterior axis?
A
- The AP axis is set out in parallel and before the formation of neuroectoderm
- The CNS is derived from the neuroectoderm
- Neuroectoderm specification is preceded by the establishment of the embryonic cranial-caudal axis
- The appearance of the primitive streak marks the future caudal end of the embryo and establishes the cranio-caudal and left-right axes —> this allows establishment of neuroectoderm
5
Q
What is the molecular control of body axes establishment in Xenopus?
A
- There are maternal signals which are initially confined to the vegetal pole which then translocate to the dorsal part of the embryo
- This induces the accumulation of nuclear B-catenin
- This activates the genes required for the organiser function
- The organiser becomes a source of signals such as Nodal, Chordin and Noggin (inhibit serine/threonine kinase pathway), that establish the axes of the developing embryo
- They are antagonists for BMP pathway (serine/threonine pathway inhibition)
- All these signals are secreted and diffuses through the embryo, generating gradients
-These signals are also essential for neuroectoderm specification
6
Q
What is the molecular control of the AP axis establishment in mouse?
A
- Amniotes are tetrapods vertebraes including reptiles and mammals
- The topology of the amniote embryo is ‘inside-out’, with the embryo being cylindrical
- Just prior to gastrulation, the embryo is formed by the epiblast surrounded by a layer of cells called the visceral endoderm/hypoblast
- The first indication of axis is establishment of distal visceral endoderm (DVE) at the distal part of visceral endoderm
- DVE expresses Lefty1 at its moment of specification
- Cells from DVE migrate from proximally and they recruit other cells that also start expressing Lefty1 (antagonist TGFB superfamily/Nodal - serine/threonine) and also DKK1 (antagonist of Wnt pathway)
- These cells migrate to the anterior visceral endoderm (AVE) - this is the future anterior region of embryo
- Hence as a result of AVE secreting Lefty1 and DKK1, there is repression of Wnt and Nodal in the surrounding tissue. As a result, Wnt and Nodal activity are confined to the opposite pole (posterior edge) which induces the formation of the primitive streak
- It’s important to mention that the first indication of the AP axis in the mouse embryo is the presence of AVE, not primitive streak.
- AVE is a reliable landmark for anterior pole
-BMP is expressed in the most proximal part of the developing embryo throughout the formation of DVE all the way to the primitive streak formation
- BMP is very important for DVE specification
- BMP facilitates Wnt and Nodal for specification of primitive streak at the posterior edge
- This early BMP4 activity promotes Nodal but represses Lefty1
- Nodal induces Lefty1 but BMP inhibits Lefty1
- This regulatory interaction leads to Lefty1 expression in the most distal portion of the visceral endoderm
7
Q
What is neural induction in Xenopus and fish?
A
- Organiser is in the dorsal portion —> this the source of signals: chordin, noggin, follistatin (these are secreted antagonists of BMPs)
- BMPs are expressed in all ectoderm of developing embryonic
- BMP induce the formation of epidermis from ectoderm
- Chordin, Noggin, Follistatin repress BMP which leads to specification of neural fate in the region next to the organiser
- This is called the ‘default model’ of neural induction because the ectoderm defaults to an epidermal fate
- Neural induction requires antagonism of BMP4 by chordin, noggin and follistatin
- This is molecularly conserved from flies all the way up to mammals
8
Q
What is neural induction in the mouse?
A
-Neural induction in the mouse also requires BMP antagonism
- A region of epiblast (distal) are specified as neuroectoderm
- This requires antagonism of BMP activity by chordin released from the gastral organiser (MGO) at the tip of the primitive streak
- This prevents the distal region of the epiblast being confined to epidermal fate due to BMP activity
9
Q
What is neural induction in humans?
A
-The molecular signals from earlier are conserved
- The human embryo is flat, with a primitive streak extending from the caudal end to roster all end as gastrulation progresses
- When it reaches the rostral end, it starts to regress and lays down a structure called the notochord just below the ectoderm
- Notocord process (tip of notochord) and primitive streak are sources of neural inducers (BMP antagonists - chordin, noggin)
10
Q
What are the responsibilities of the vertebrae ectoderm?
A
- The vertebrae ectoderm has 3 major responsibilities
1. One part of ectoderm becomes neural plate (presumptive neural tissue induced by prechordal plate and notochord). The neural plate involutes into the body to form the neural tube (precursor of CNS)
2. One part forms the epidermis
3. Presumptive neural crest is found between the compartments forming the epidermis and CNS. Neural crest cells migrate away to generate PNS, melanocytes and parts of the heart
11
Q
What is ectoderm specification?
A
- Ectoderm specification occurs during gastrulation through regulation of BMP levels in ectodermal cells
- High levels of BMP specify ectodermal cells to become epidermis
- Intermediate levels of BMP specify ectodermal cells to become neural crest cells
- Low levels of BMP specify ectodermal cells to become neural plate (neuroectoderm)
- The processes by which the 3 ectodermal regions are made are distinct from each other - this is neurulation
- Embryo undergoing neurulation is called neurulation
- Neurulation directly follow gastrulation
12
Q
What is Sox expression in the Neural plate?
A
- Cells of neural plate are characterised by expression of Sox family of transcription factors
- These factors activate genes that specify the cells to become the neural plate and inhibit the formation of epidermis and neural crest cells by blocking the transcription and signalling of BMPs
-The expression of Sox transcription factors establishes the neural plate and neural precursors that can form all the cell types in the CNS
13
Q
What is the neural plate?
A
- 1st stage or neurulation is formation of neural plate
- Neural plate is thickened ectoderm along dorsal midline
- Gives rise to neural tube and neural crest
- Neural plate lies on surface of embryo but the future nervous system will be inside the mature body. Hence, the neural plate has to move inside the embryo and form the neural tube
- This is done through neurulation
-There is primary and secondary neurulation
14
Q
What is primary neurulation and secondary neurulation?
A
Primary neurulation:
-Cells surrounding the neural plate direct the neural plate cells to proliferate, invaginate into the body and separate from the surface ectoderm to form a follow underlying tube
Secondary neurulation:
-Neural tube arises from aggregation of mesenchyme into a solid cord that can then form cavities which then forms a hollow tube
- Neurulation is complete when 2 separately formed tubes unite
- Size of transition zone is thoracolumbar in humans
- In many vertebrates, primary neurulation forms anterior neural tube; secondary neurulation forms posterior neural tube
- In mammals, secondary neurulation occurs at sacral level
15
Q
What is primary neurulation in detail?
A
- After formation of neural plate, its edges thicken and move upwards to form the neural folds.
- This forms a U shaped groove in the centre of the plate, diving the future left and right sides of the embryo
- The neural folds migrate towards the midline of the embryo, fusing to form the neural tube beneath the overlying ectoderm
- There are 4 stages which overlap:
1) elongation and folding of neural plate=
- cell divisions within neural plate are preferentially on the AP axis
- this fuels continued axial elongation that was associated with gastrulation
2) Folding of neural plate
- formation of hinge regions where the neural plate contacts surrounding tissues
- in birds and mammals, cells at the midline of the neural plate form the median hinge point (MHP)
- MHP cells are firmly anchored to the underlying notochord, forming a hinge which enables the creation of a furrow which is the neural groove in the dorsal midline
3) convergence of the neural folds
- after the neural groove is created, 2 dorsolateral hinge points (DLHPs) are induced by and anchored to surface ectoderm
- The plate bends around these hinge regions
- each hinge point acts as a pivot, directing the rotation of cells around it
- continued convergence of surface ectoderm pushes towards midline of the embryo, providing another force that bends the neural plate, causing the convergence of the neural folds
- This movement of the presumptive epidermis and the anchoring of the neural plate to underlying mesoderm may also be important for ensuring that the neural tube invaginates inwards, not outwards
4) Closure of neural tube
- The neural tube closes as the paired neural folds come into contact in the dorsal midline
- the folds adhere to each other and the neural and surface ectoderm cells from one side fuse with their contralateral counterparts
- During this fusion, cells at the apex of neural folds delaminates to become the neural crest cells
16
Q
How are hinge points formed and regulated?
A
Formation:
- MHP and DLHPs are 3 regions in the neural plate where epithelial cell shape changes occur
- The epithelial cells in these regions adopt a ‘wedged-shaped’ morphology along the apicobasal axis -they are wider basally than apically
- Similar to the bottle cells in gastrulation, localised contraction of actomyosin complexes at the apical border of the neuroepithelial cells of the hinge points causes apical constriction
- To facilitate this, nuclei are retained in the basal portion of cells
Regulation:
- We know that notochord induces cells of MHP to become wedge shaped
- Shh is produced in notochord and is required for the induction of floor plate cells in the neural plate, which is turn form the MHP
- In DLHP, Noggin appears to be critical for hinge formation
- In mice, loss of Noggin results in severe failure of neural tube closure
- Noggin is expressed in the neural folds and this expression alone is sufficient to induce DLHPs
- Noggin is also expressed in notochord
-Shh is produced by notochord which prevents ectopic hinges from forming
17
Q
What is the role of BMP for hinge point formation?
A
- BMP inhibits hinge joint formation
- BMP is expressed by dorsal surface of ectoderm
- Noggin is an antagonist so it may seem reasonable to think that BMP inhibition results in changes in cell shape that lead to DLHP
- Experiments have shown that when BMP is constitutionally active, it binds to its receptors and prevents hinge joint formation; when BMP is completely repressed, there is ectopic and exaggerated MHP and DLHP formation
- This shows that intermediate levels of BMP is required for normal hinge formation. Neural plate cells at this level of BMP will undergo apical constriction and basal thickening - this occurs through a modification of the recruitment of proteins that stabilise junctions proteins and maintain size equality between the apical and basal membrane, preventing folding
- Lack of BMP (caused by Noggin) leads to relaxation of these junctions allowing apically restricted actomyosin contractions and a shortening of apical membrane
-Shh from notochordal plate prevents ectopic hinges from forming in the neural plate
18
Q
How does the neural tube close?
A
- Closure of neural tube doesn’t occur simultaneously
- Best in amniotes whose body axis is elongated prior to gastrulation
- Neural tube closure in mammals is initiated at several points along AP axis (5 sites)
- Rostral closure site (1) is located at the junction between the spinal cord and hindbrain. It closes by bidirectional zipping of neural folds
- Closure site 2= midbrain/forebrain boundary. Zipper like mechanism and dynamic cell extension occurs
- Closure site 3= rostral forebrain. The DLHPs appear to be fully responsible for neural tube closure
19
Q
How does the neural folds zip up?
A
- During DLHP bending, dynamic cell processes extend from the tips of the neural folds
- Inhibition of myosin prevents zipper advancement
20
Q
What is fusion and separation of neural tube?
A
- The neural tube eventually forms a close cylinder that separates from the surface ectoderm
- This separation is mediated by expression of different cell adhesion molecules
- Neuroectodermal cells initially express E-Cadherin, though they stop expressing it as the neural tube forms
- They switch to N-Cadherin instead
- This means the surface ectoderm and neural tube tissues no longer adhere to each other
- The Grainyhead transcription factors are important to switch was E-cadherins to N-cadherins
-Mice with mutated Grainyhead-like2 and 3 have severe neural tube defects including exencephaly and spina bifida
21
Q
What are neural tube defects?
A
- Failure of closure of posterior neuropore results in spina bifida - the severity depends on how much of the spinal cord remains exposed
- Failure to close sites 2 and 3 in the rostral neural tube keep the anterior neurones open, resulting in anencephaly which is usually lethal
- In anencephaly, the forebrain remains in contact with the amniotic fluid and subsequently degenerates. The fetal forebrain ceases development and the cranial vault fails to form
-Failure of the entire neural tube to close causes craniorachischisis
22
Q
What factors affect neural tube closure?
A
- Can result from both genetic and environmental causes
- More than 300 genes play a role in neurulation
- Mutations in genes like Pax3, Shh, Grainyhead, Tfap2 and Openbrain show that these genes are essential for the formation of the mammalian neural tube
- Environmental factors like drugs, cholesterol and folate, diabetes, obesity and toxins can all affect neural tube closure
23
Q
What is the role of folate?
A
- The early use of folic acid antagonists led to fetuses with neural tube defect
- Since then, many trials have shown clear correlations of neural tube defects with folic acid deficiency
- Folic acid is recommended for pregnant women
- Folic acid is important nutrient for regulating DNA synthesis during cell division in the brain and is critical in regulating DNA methylation
- Epigenetic mechanisms are essential for proper neural tube development
24
Q
What is secondary neurulation?
A
-Secondary neurulation takes place in the most posterior region of embryo during tailbud elongation
- It involves production of mesenchymal cells from the prospective ectoderm and mesoderm, followed by condensation of these cells into a medullary cord beneath the surface ectoderm
- After mesenchymal to epithelial transition, the central portion of the medullary cord undergoes cavitation to form several lumens
- The lumens then coalesce into a single central cavity
Control of secondary neurulation:
- After Hensen’s node has migrated to the posterior end of embryo, the caudal region of the epiblast contains a precursor cell population that gives rise to both neural ectoderm and paraxial (somitic) mesoderm as the embryo’s trunk elongates
- The ectodermal cells that will form the posterior (secondary) neural tube express the Sox2 gene, whereas the ingressing mesodermal cells (which no longer encounter high levels or BMP as they migrate beneath the epiblast) don’t express Sox2.
- Instead, the ingressing mesodermal cells express Tbx6 and form somites
- Tbx6 transcription factor represses neural-inducing Sox2, preventing too many neural tubes from forming
-During primary neurulation, the surface ectoderm and neural ectoderm are intimately connected through the process of neural tube closure and fusion, whereas in secondary neurulation, these 2 tissues are essentially uncoupled and develop independently of each other
25
What is the transition zone?
- In human and chick embryos, there appears to be a transitional region at the junction of the anterior (primary) and posterior (secondary) neural tubes
- Neural tube formation in this transition zone is called Junctional neurulation
- In human embryos, coalescing cavities are seen in the transitional region, but the neural tube also forms by the bending of neural plate cells
- The junctional neural tube in the chick is a mosaic of both ventral mesenchyme cells and dorsal neural ectodermal cells
- In addition to directly providing dorsal epithelial cells to the junctional neural tube, neural plate cells also undergo an epithelial to mesenchyme transition and ingress into the underlying mesenchyme pool
26
What is anteroposterior axis?
- The early mammalian neural tube is a straight structure
- Before the posterior portion of the tube has formed, the anterior portion is undergoing drastic changes
-It balloons 3 primary vesicles along the AP axis: the forebrain (prosencephalon), midbrain (mesencephalon) and hindbrain (prosencephalon). These are subdivisions of the neural tube
- The forebrain forms the cerebral hemispheres
- Neurones of midbrain go on to be involved in motivation, movement and depression
- Hindbrain becomes cerebellum, pons and medulla
- By the time the posterior neural tube closes, secondary vesicles have formed
- The forebrain becomes the telencephalon (which gives rise to the hemispheres) and diencephalon (forms the optic vesicle)
- The midbrain gives rise to mature midbrain
- Hindbrain gives rise to metencephalon (gives rise to cerebellum and pons) and the myelencephalon (gives rise to medulla)
27
What is Rhombencephalon?
- Rhombencephalon develops as a segmental pattern that specifies where certain nerves originate
- Rhombomeres (periodic swellings) divide the Rhombencephalon into smaller compartments
- Each rhombomere express a unique combination of transcription factors, generating a rhombomere specific latter of neuronal differentiation
- As a result, each rhombomere produces different fated neurones
- Neural crest cells derived from rhombomeres go on to form ganglia, with each rhombomere ganglion producing a different type of nerve
- The AP patterning of hindbrain and spinal cord is controlled by a series of genes that include HOX gene complexes
28
What is the dorsoventral axis?
- The neural tube is polarised along its dorsal-ventral axis
- In the spinal cord, the dorsal region is the place where the spinal neurones receive input from sensory neurones
- Ventral region is where the motor neurones reside
- In the middle are numerous interneurones that relay information between the sensory and motor neurones
- These differentiated cell types organised along the dorsoventral axis arise from progenitor cell populations located adjacent to the ventricles, which run along the AP axis
- Each progenitor domain can be defined by its expression of specific transcription factors (such as products of HOX genes) which specify cells to differentiate into specific classes of neuronal and glial cells that make up the CNS
- The dorsal-ventral polarity of the neural tube is induced by morphogenetic signals coming from its immediate environment
- The ventral pattern is imposed by the notochord, whereas the dorsal pattern is induced by the overlying epidermis
- Specification of the axis is initiated by 2 major paracrine factors - Shh from the notochord and TGFB from dorsal ectoderm
29
What is the role of Shh?
- Shh secreted from the notochord induces the MHP cells to become the floor plate of the neural tube
- The floor plate cells also secrete Shh, which forms a gradient that is highest in the most ventral portion of the neural tube
- Cells experiencing the highest concentrations of Shh develop into motor neurone progenitors and a class of interneurones called V3 neurones
- Moderate and lower levels of Shh induce increasingly more dorsal progenitor populations
30
What is the role of TGFB?
- The dorsal fates of the neural tube are established by proteins of the TGFB superfamily, especially BMP4, BMP7, dorsalin and activin
- Initially, BMP4 and BMP7 are found in epidermis
- Just as the notochord establishes a secondary signalling centre on the ventral side of the neural tube (the floor plate cells), the epidermis establishes are secondary signalling centre by inducing BMP4 expression in the roof plate cells of the neural tube
- The BMP4 protein from the roof plate induces a cascade of TGF-B proteins in adjacent cells
- Dorsal sets of cells are therefore exposed to higher concentrations of TGFB proteins and at earlier times when compared with the more ventral neural cells
31
What is progenitor identity?
- Progenitor identity is determined by the unique gene regulatory network it expresses
- The system of differential gene expression a cell exhibits is dependent on the combination of its distance from, and duration of, exposure to the morphogenetic signalling centres
- Cells adjacent to the floor plate that receive high concentrations of Shh synthesise the transcription factors Nkx6.1 and Nkx2.2 and become the ventral (V3) interneurones
- The cells dorsal to them, exposed to slightly less Shh (and slightly more TGFB), produce Pax6 and Olig2 and become motor neurones
- The next 2 groups of cells, receiving progressively less Shh, express Pax6 alone and become the V2 and V1 interneurones
- Finally, the cells at the dorsal most segment of the neural tube express Pax7 and become dorsal progenitors
32
What is the gene regulatory network?
-It had been thought that the intersecting gradients of Shh and TGFB signals would be sufficient to instruct the synthesis of the various transcription factors but the regulatory network is far more complex, and appears to integrate both spatial and temporal distributions of the morphogen signalling
- If Pax7-expressing intermediate neural tube explants are exposed to increasing concentrations of Shh, they will stop expressing Pax7 and will express Olig2 and Nkx2.2 in a dose dependent manner
- If these same explants are exposed to a constant concentration of Shh over an extended period of time, they first express Olig2, followed by increasing levels of Nkx2.2
33
How is Shh signalling involved?
-In vertebrates, the main downstream effectors of Shh signalling are the GLI family of transcription factors
-They function as repressors or activators based on the absence or presence of Shh
-Hence, Shh from the notochord and floor plate is transduced
into a ventral-to-dorsal gradient of GLI activators to GLI repressors
- An early expansion of GLI activator function coincided with the initial induction of the broad and overlapping expression of progenitor cell transcription factors, yet GLI activity was not maintained over the course of cell differentiation in the neural tube
- Despite this reduction in Shh signalling over time, the domains of the progenitor specific transcription factors still became highly refined
34
What is transcriptional cross-repression?
- Gene regulatory networks play a direct role in reinforcing, refining and maintaining progenitor cell fates through the mechanism of transcriptional cross-repression, whereby transcription factors repress each other
- Gain and loss of function manipulations of transcription factors in progenitor cells have demonstrated that different transcription factors such as Olig2 and Nkx2.2 (which are expressed in adjacent domains), can mutually repress each other’s expression, thereby helping define the borders between adjacent
- Transcriptional cross-repression integrated into a model that includes Shh provides a mechanism for a cell to ‘remember’ the Shh signal and consequently, its position in the neural tube
- At the earliest time of induction, Shh from notochord induces Gli in the floor plate cells
- This action isn’t sufficient to activate Olig2 or Nkx2.2 or repress Pax6
- As development increases, GLI is able to induce Olig2 which inhibits Nkx2.2 and Pax6
- As the most ventral cells experience higher concentrations of Shh for longer periods, Nkx2.2 is activated and suppresses Olig2
- This pattern can be retained even when GLI levels decrease
35
How do all the axes come together?
- The model of dorsal-ventral patterning by TGF-B and Shh morphogens relates to cell fates throughout three CNS along the rostral-caudal axis
- But there are differences in how the anterior and posterior regions of the neural tubes from, which are defined by primary and secondary neurulation
- Progenitor cells in the anterior regions of the neural tube (which become the brain and most of spinal cord) adopt a proneural fate directly from the epiblast
- Cells in the posterior begin as bipotential neuromesodermal progenitors (NMP) that undergo a transition to become neural or somitic cell types
- The neural cells go on to form the caudal end of the neural tube
- NMPs are born in the caudal lateral epiblast during tailbud elongation and are positively maintained by Fgf8 and Wnt signals
- It is these antagonistic gradients of retinoic acid and Fgf/Wnt along the rostral-caudal axis that establish a ‘road’ to NMP maturation
- An NMP cell is born into the tailbud and enters the mesenchyme (undergoing secondary neurulation)
- NMP cells that enter neural mesenchyme become the prenatal progenitor cells and are initially competent to respond to either Shh or BMP signals by differentiating into either floor plate or roof plate respectively
- As the tailbud elongates, these preneural NMP cells become positioned further from Fgf/Wnt and closer to retinoic acid
- This repositioning triggers a switch in their competency to respond to Shh/TGFB signals, thus allowing patterning of the proneural progenitors along the dorsoventral axis of the maturing neural tube
36
What are the environmental factors associated to NTDs?
- Maternal diet it really important
- Neurulation occurs at 3-4 weeks of embryogenesis - most women don’t even know they’re pregnant yet
- Vitamin deficiencies (folate and inositol) are often associated with NTDs
- High sugar levels are also associated with NTDs
- Maternal obesity and diabetes also contribute to NTDs
- Teratogenic agents (e.g valporate - an epilepsy medication) are associated with NTDs
37
What is the association of Folate and NTDs?
-Clinical trials in humans in the 1990s showed that maternal folic acid supplementation prior to and during pregnancy had a preventive effect against NTDs (helps with DNA synthesis)
- Folate supplementation is the only known intervention that prevents any type of congenital anomaly
- There are suggestions that folate also reduces the incidence of palate and heart defects
-There are still some folate resistant NTD
