Neurulation 2 Flashcards
How is the anterior-posterior axis established in Xenopus?
Xenopus: At the early stages of development, there are maternal signals deposited which leads to deposition of nuclear beta-catenin in the vegetal pole and is confined there. The beta-catenin is later translocated to the dorsal part of the embryo, whereby there is an accumulation of nuclear beta-catenin. Beta-catenin in this region activates genes needed for organiser functions. The organiser will become a source of signals that establishes the axes for the developing embryo and neuroectoderm specification. These signals include Nodal (which is an agonist of the TGF-family), chordin and Noggin (antagonists of TGF-family). They spread throughout the embryo creating gradients, especially chordin and Noggin.
What is the molecular mechanism for the establishment of the anterior-posterior aixs?
Prior to gastrulation, the epiblast domain is surrounded by the visceral endoderm. The first indication of axes is the specification of a structure called distal visceral endoderm (DVE) which is established at the distal end of the visceral endoderm (BMP plays a role in its specification). It produces a molecule called Lefty 1 (antagonist of nodal so it inhibits TGF-family). The cells of DVE migrate proximally at one pole and migrate other cells that express Lefty1; they also start to express Dkk1 (inhibits Wnt pathway) – these form the anterior visceral endoderm (AVE). AVE is a source of 2 molecules: Lefty1 and Dkk1. Lefty1 and Dkk1 repress Wnt and Nodal in surrounding tissues, so as a result, Wnt and Nodal activity will be confined to the opposite pole of the developing embryo. Wnt and Nodal will induce the formation of primitive streak at the posterior visceral endoderm, along with BMP.
BMP is important for the specification of the DVE and works together with Wnt and Nodal in the specification of the primitive streak. The early BMP4 induces Nodal which induces Lefty1; BMP inhibits Lefty1 (around the sides of the DVE), hence, this leads to activation of Lefty1 only in the most distal portion of the developing embryo.
What is neural induction?
In the Xenopus embryo, the organiser is specified in the dorsal portion of the embryo and is a source of signals such as chordin, Noggin and Follistatin – they all antagonise the activity of BMPs. BMPs are expressed in the whole ectoderm and they usually induce the formation of the epidermis in the ectoderm. However, due to chordin, Noggin and Follistatin inhibiting BMP, it leads to the specification of the neural fate of the ectoderm in this region around the organiser. The process of BMP antagonism is conserved in the neural induction of every organism.
In humans, the primitive streak progresses from caudal to rostral then it starts regressing. As it regresses, the notochord (which is below the neuroectoderm) is established. The notochordal process and the primitive node are the sources of chordin and Noggin, to inhibit BMP to allow neural fate to be established.
What is neural tube closure?
So now the neural plate has been specified and it is organised as a flat layer along the dorsal portion of the embryo. Initially, the neural plate is found on the ectoderm but eventually has to be buried underneath the ectoderm and fold over itself to form the neural tube – this is neural tube closure.
In order for the neural plate to fold over itself, the edges of the neural plate have to elevate and become pushed next to each other in order to fuse. Once they have fused, it detaches from the overlying ectoderm and becomes buried within the embryo.
In Xenopus, this closure process occurs simultaneously along the AP axis.
In humans, it starts in specific regions and progresses anteriorly and posteriorly. Defects in neural tube closure can lead to many neural tube defects whereby the neural tube is exposed to the extracellular medium.
What are the different points of closure?
There are different points where the neural tube fuses called closure points.
Closure 1 is the first one that triggers closure of the neural tube and is located at the edge between the hindbrain and spinal cord, and it progresses anteriorly and posteriorly.
Closure 2 is at the edge between the forebrain and midbrain and will progress anteriorly and posteriorly; it will also generate closure 3 which is at the most rostral part of the neural plate.
In humans, there are more closure points such as closure 4 which is in the hindbrain; closure 5 which is in the most caudal region. This happens by primary neurulation – the neural plate that gives rise to the nervous system, enclosed by primary neurulation
What is primary neurulation?
Primary neurulation involves rolling up of sheet of cells to form a tube; closure is initiated by folding and ‘zipping up’.
The closure at the cranial and caudal neuropores are the last regions to become fused. So essentially, the neural plate is folded and the edges of the neural plate are elevated so that they come together and fuse and detach from the overlying ectoderm.
Steps: the neural plate needs to be shaped, so it elongates and narrows – this brings the edges of the neural plate closer together and is crucial for closure of neural tube.
The neural plate folds at different points.
The first folding point occurs at a point called the median hinge joint which runs along the midline of the neural plate along the AP. It will lead to the sinking of the neural plate underneath the ectoderm, bringing together the edges of the neural plates. Then there is the elevation of the edges of the neural plate so that they can come closer together. There is the convergence of the neural plate edges and eventually oppose each other. This is helped by the formation of hinge points in the dorsolateral walls of the folding neural tube.
Eventually, the edges of the neural tube come together and start to fuse.
Then there is migration of neural crest cells which give rise to variety of cell types that spread throughout the body. Simultaneously to the migration of the neural crest cells, the 2 opposing sides of the neural tube get really close and form a tube underneath the epidermis (ectoderm).
-Key (molecular mechanism): Shaping of the neural tube occurs by convergence and extension which is driven by the planar cell polarity pathway (PCP) which is the non-canonical Wnt pathway. Tube formation involves bending at hinge points and fusion of edges of the neural tube. The median hinge point is induced by signals released by notochord (it is anchored to notochord). The dorsolateral hinge points are anchored to the ectoderm and also dependent on signalling mechanisms. Cell wedging at hinge points is driven by microtubules and actin filaments.
What is secondary neurulation?
Secondary neurulation occurs in the most caudal region (in the tailbud). A group of mesenchymal and ectodermal cells become condensed to form a rod (which is initially NOT hollow) and it later becomes hollow and forms a tube through mesenchymal to epithelial transition.
-The 2 portions of the neural tube (where primary and secondary neurulation has occurred) become fused and this occurs in humans at the levels of somite 30-31, at the lower back. Defect in fusion between the 2 components of the neural tube can lead to spina bifida.
What is the detailed molecular process for primary neurulation?
Wnt will interact with Frizzled; there are a number of coreceptors working in this pathway which call lead to activation of Dishevelled (Dvl), which in turn leads to activation of a number of downstream events in the cells which can involve transcription and cytoskeletal regulation. Mutations in proteins involved in the PCP pathway leads to neural tube defects. It can cause neural plate to become abnormally broad with a non-bending region between neural folds, leading to chraniorachichisis (complete opening of neural tube so that the whole neural tube is exposed to the extra embryonic medium).
Before convergence and extension of neural tube, we have folding of the neural plate. This folding occurs in specific regions of neural plate. The first folding occurs along the midline of the neural plate called the medial hinge joint (at this point, the cells change shape to bottle shaped) – this change in cell shape is driven by cortical cytoskeleton on apical surface of cells as the apical surface constricts. This happens along the mediolateral axis of the cell. This is controlled by Wnt-PCP pathway whereby this pathway controls the polarised contraction of the actomyosin cytoskeleton at the apical cortex of these cells; this drives the constriction along the mediolateral axis.
Cell wedging occurs at specific regions of the neural plate and varies along the anterior-posterior axis. In the upper spine, we only have the median hinge point, whereas in the intermediate spine, we start seeing the dorsolateral hinge points as well. In the lower spine, you can see the median hinge point is less apparent than dorsolateral hinge point. This occurs due to bending a cell sheet (wedging of individual cells by contraction of their apical ends driven by myosin motor proteins acting on the actin filament).
The position of the hinge points are controlled by Shh and MBP pathways. Shh is produced by notochord leads to establishment of median hinge point and inhibits dorsal lateral hinge joint. In embryos that don’t have Shh due to lack of notochord, there will be no establishment of median hinge point, but there will be a dorsolateral hinge point all along the whole-body axis. BMP2 is secreted from dorsal surface of ectoderm and inhibits dorsal lateral hinge point formation in dorsal portion of neural plate. Noggin (BMP antagonist) is also released which prevents this inhibition of dorsolateral hinge point, allow dorsolateral hinge point formation. If Shh concentration is high enough, it can repress Noggin expression; so lack of Noggin means more BMP available to repress the formation of the dorsal portion of the neural plate. In the upper spine, Shh is secreted strongly from the notochord so suppresses Noggin so there is more inhibition of dorsolateral hinge point formation. In low spine, notochordal Shh is diminished so Noggin is not repressed so it can cause dorsolateral hinge point formation.
Neural crest cell migration occurs prior to closure in the cranial neural tube but after closure in the spinal region, the migration of neural crest cells from the anterior portion of the neural tube seems to be important for accurate closure.
As the neural tube closes, the neural crest cells at the edges of the neural plate migrate away from the edges of the folding neural tube and spread through the embryo to give rise to diverse pool of cell types.
Ectoderm generates forces to contribute to closure of neural tube. Furthermore, apoptosis is also required in the most anterior portion of the neural tube.
What is neural tube patterning?
AP and dorsoventral patterning are established concomitant to neural induction and neural tube closure.
So as the neural tube folds and close, the AP and DV are established.
At the end of the closure, the different regions that will prefigure the different structures of the differentiated nervous system become specified.
How is the AP axis established?
The organiser at the future dorsal portion secretes molecules important to neuroectoderm specification. The embryo and neuroectoderm become elongated; the organiser and the surrounding tissues are taken farther away from the most anterior portion of the neuroectoderm.
The organiser and the mesoderm that starts invaginating along the organiser are sources of additional signals that posteriorize the neuroectoderm – these signals are from families FgFs, retinoic acid and Wnts. It creates a gradient along the AP axis (higher concentration posteriorly; low levels anteriorly) –> this ensures the anterior aspect doesn’t get the posteriorizing signal (the anterior neuroectoderm is protected from posteriorizing signals).
Elongation of the AP axis takes the anterior portion of the neuroectoderm away from the source of posteriorizing signals.
Furthermore, there are antagonists produced by tissues underlying the most anterior portion of the neuroectoderm which protect the anterior portion from the Fgs, retinoic acid and Wnts –> these allow anterior portion to acquire anterior neuroectoderm character while the posterior portion will acquire posterior fates.
The initial rough patterning of the neuroectoderm leads to the formation of the main subdivisions of the neural tube along its AP axis. These are 3 main brain vesicles along the AP axis. These subdivisions are mesencephalon (midbrain), prosencephalon (forebrain) and rhombencephalon (hindbrain). The spinal cord will be found posterior to the hindbrain. These vesicles become further subdivided into secondary vesicles as the pattern becomes refined and eventually the secondary vesicles will give rise to different derivatives.
Once the AP of the neural tube is established, the patterns are further refined by signals from secondary signalling centres within the neural tube. The difference between primary signalling centres and secondary signalling centres is that primary signalling centres affect the whole embryo and have critical roles early on for the patterning of the embryo; secondary signalling centres refine the initial patterning and have more local effects around tissues that surround them.
These secondary organisers are established due to the AP axis. These secondary organisers create produce morphogens that create morphogen gradients and confer positional information around the signalling centres. They act as morphogens. The signals confer different fates at different concentrations at the neuroectoderm and different genes will start being expressed
What are the secondary organisers?
- Anterior neural boundary (ANB) which are cells along the anterior edge of the neural tube which release FGF8, Wnt and antagonists
- Zona limitans intrathalamica which is in the diencephalon; it releases Shh
- Roof plate produces Wnt
- Midbrain-hindbrain boundary which is found posteriorly between midbrain and hindbrain; releases FGFs and Wnt
- BMP is found on the outside of the roof plate which contributes to patterning of dorsal portion of neural tube
How is the midbrain-hindbrain boundary established?
The position of the midbrain-hindbrain boundary is prefigured by the transcription factors Otx2/Gbx1 interface.
Otx2 is expressed in the whole portion of the anterior portion of the neuroectoderm and covers the midbrain. GBX1 will be expressed in the future hindbrain. The expression of these 2 genes is a consequence of the AP patterning (mainly due to Wnt activity is higher in the posterior portions of the neuroectoderm; lower Wnt in anterior portions during neuroectoderm patterning)
So, the Wnt function establishes Otx2/Gbx1 interface. High levels of Wnt induces GBX1 and repress Otx2. The midbrain-hindbrain boundary arises at the boundary between Otx2 and Gbx1 expression.
Cells along the midbrain-hindbrain boundary secrete Wnts and Fgs which create gradients where the highest conc is at the midbrain-hindbrain boundary, lower concentrations anteriorly and posteriorly. This will influence the tissue around it to lead to the formation of the midbrain and cerebellum. This shows that the same signals released by the midbrain-hindbrain boundary give rise to different fates anteriorly and posteriorly because the different cells express Otx2 or Gbx1.
What is dorsal-ventral patterning of the neural tube?
Dorsal-ventral patterning requires Shh activity. Shh is released by the notochord which is the ventral to the neural tube, and it induces the floor plate (which is the ventral most portion of the neural plate).
The floor plate also releases Shh. The concentration of Shh will be higher in the ventral region than in the dorsal. This gradient confers different fates along the axis so each domain can give rise to different types of neurons when the neural plate starts to differentiate.
A combination of Shh, TGF-B and Wnt signals are required to pattern the dorsal portion of the neural tube. BMP concentration is greater in the dorsal aspect than the ventral aspect – so BMP patterns the dorsal neural tube. Wnt is produced from the dorsal region which refines the levels of the activation of the Hh signalling pathway.
What environmental factors can cause neural tube defects?
Neural tube defects can be caused by vitamin deficiency e.g., folate and inositol, high levels of sugar, maternal obesity, diabetes, teratogenic agents. Mums should take folate supplements
What is holoprosencephaly?
Holoprosencephaly (HPE): a defect of dorso-ventral forebrain patterning HPE is a structural malformation of the forebrain characterised by impaired midline cleavage of the brain. In humans, this process occurs between weeks 3 and 4 of embryonic development. HPE can be classified as alobar, semilobar or lobar, depending on the degree of severity of the midline defect.
- Alobar HPE refers to the formation of a single ventricle, and the absence of interhemispheric fissure. Not only the brain, but also other CNS derivatives and face structures are affected in this from. For example, the separation of the eye field into two optic primordia does not occur, and the affected individuals also display cyclopia.
- Semi lobar HPE shows partial cortical separation, rudimentary hemispheres and a single ventricle.
- Lobar HPE shows separate ventricles, but there is still incomplete frontal cortical separation.
- Some milder forms of HPE have been described, which show much milder midline defects, sometimes only identifiable by a single maxillary median incisor or hypotelorism (close set eyes), and no brain malformation (microforms of HPE).
HPE is very heterogeneous, but one of the main signalling pathways that has clearly been linked to this disease is the SHh signalling pathway. Mutation in SHH PTCH1 GLI2 CDON and some other genes functionally associated to the HH pathway in the brain, such as ZIC2 and SIX3 have been found linked to HPE. Shh is expressed in the embryonic ventral midline, underlying the developing neural plate, as is essential for the specification of the ventral structures of the neural tube. In the most anterior portion of the neural plate, absence of Shh function leads to the misspecification of the future ventral regions of the forebrain. The separation of the dorsal wings of the forebrain into two hemispheres, and the separation of the eye field to give rise to two optic vesicles, require the specification of these ventral structures, and do not occur in their absence.
