Following gastrulation, organogenesis (formation of organs) begins and part of this is neurulation, the formation of the nervous system.
In insects, neuroblasts ingress individually to form the central and peripheral nervous system. In vertebrates, neurulation begins with the phylotypic state, where only the neural tube formation (from which the spinal cord arises) has occurred and all vertebrate embryos look near-identical; later, distinguishing features like fins, legs, wings, etc arise.
Neurulaion in Insects
In invertebrates, neural precursors undergo an epithelial-to-mesenchymal transition by ingresssing individually from the neuroectoderm (a specialized region of ectoderm) into the embryo interior.
Ingressing cells constrict at their apical surfaces via microfilaments and elongate via microtubules, while losing their tight junctions and adherens junctions. Once in the interior, these neural precursors proliferate and are known as neuroblasts.
Neuroectoderm in Drosophila consists of two lateral stripes on either side of the embryo, arises during the dorsal-ventral patterning system and gives rise to epithelium and neuroblasts. Cellular commitment in insect embryos to a neural fate is autonomous (not involving signaling from neighboring cells) and is based only on the cell's interpretation of its position in the dorsal-ventral axis. These neuroblasts are determined in two steps, described immediately below.
|1st Step||Groups of cells (proneural clusters) within the neuroectoderm begin to express proneural genes (a family of bHLH transcription factors encoded by the Achaete-scute complex) and thus become competent to form neuroblasts. Neuroblasts seldom form in Achete-scute-/- mutants.|
|2nd Step||A cell that has ingressed from the proneural cluster will inhibit remaining cells in that cluster from becoming neuroblasts. The genes involves are neurogenic genes; the entire neuroectoderm of neurogenic-/- mutants gives rise to neuroblasts.|
Notch is a critical member of the neurogenic genes describes above.The Notch protein is a transmembrane receptor with multiple epidermal growth factor (EGF) repeats in its extracellular domain. Other neurogenic genes encode ligands (such as Delta) that bind Notch. Notch and its ligands have important roles toward cell fate determination not just in Drosophila, but in vertebrates as well.
Neurulation in Vertebrates
In vertebrates, neural precursors comprise the entire neuroectoderm.
These cells elongate along the anterior-posterior axis via convergent extension and become columnar (similar to involution and invagination during gastrulation) and appear on the dorsal side of the embryo as a raised plate of cells called the neural plate. While continuing to extend along the anterior-posterior axis, the neural plate invaginates to become the neural tube (which closes via apical constriction via microfilaments).
However, the far lateral region of the neural plate does not invaginate and instead becomes the neural crest. Starting just dorsal of the neural tube, cells of the neural crest migrate through the entire body to form the sensory and autonomous nervous system, as well as non-neural cells such as melanocytes.
The neural tube gives rise to the central nervous system: the brain at the anterior; the spinal cord at the posterior. The neural tube has three polarities:
|Anterior-Posterior||Corresponding to the A/P axis of the body, and likely determined by homeodomain proteins homologous to Drosophila patterning genes.|
|Dorsal-Ventral||Corresponding to the D/V axis of the body, and defined by Sonic hedgehog in the notochord and floorplate. Along the dorsoventral axis of the spinal cord: the most ventral region contains the floor plate, which guides axons; the dorsal region contains somatic motor neuron cell bodies (which send out axons to innervate muscles), followed by cell bodies of association neurons against the floor plate. The notochord induces the neural plate and guides its dorsoventral patterning. Transplanting an additional notochord underneath a closing chick embryo neural tube causes induction of an additional floorplate with adjacent motor neurons. Removing the notochord results in a non-functional floor plate and a lack of motor neurons.|
|Apical-Basal||Corresponding to the lumen (apical) and the surrounding basal lamina. Unclear how the A/B axis is defined. Retained in the differentiated spinal cord, there are three neural tube zones in the apical-basal direction. The most apical (facing the neural tube lumen) zone is the ventricular zone where neuroblasts undergo cell division (3H-thymidine is thus incorporated here). Cells which have undergone their last division (postmitotic) migrate from the ventricular zone to form the mantle (intermediate) zone. Finally, the outer layer contains axon projections and is called the marginal zone.|
Does the neural plate have properties which innately cause lead to neurulation? Similar to a gastrulating sea urchin vegetal plate's ability to fold on its own, an isolated amphibian neural plate will continue to round up and form a neural tube (albeit in the wrong direction). Microtubules are critical to columnarization of neural plate cells, evidenced by presences of tubules parallel to the long axis and by colchine's ability to inhibit elongation.
In vertebrates, commitment to a neural fate is based on inductive interaction with adjacent notochord tissue. In 1916, Hans Spemann wondered about the timing of tissue determination. He transplanted tissues between light and dark newt embryos at different stages of gastrulation. Spemann found that neural ectoderm is not determined in the early gastrula stage, but is determined by the late gastrula stage; thus, neural determination occurs during gastrulation. However, neural differentiation does not occur until later.
Deleting and transplanting notochord and floorplate (ventral part of neural tube) has revealed that these tissues are inducers with ventralizing effects on the neural tube. Notochord tissue specifically causes neural tube tissue to differentiate into a floorplate. Notochord and floorplate tissue both induce adjacent somite tissue to become sclerotome, the precursor of vertebrae and ribs.
In humans, defects in neural tube closure can cause various neural tube and vertebral defects, collectively known as spina bifida. Failure to close the neural tube at the most anterior position results in anencephaly (absence of the brain). By unknown pathways, increasing folic acid in the maternal diet reduces the occurrence of spina bifida.<
Dorsal-ventral patterning defines the neurectoderm in insects. In contrast, the neural plate in vertebrates is induced by the chordamesoderm (the mesoderm that will form the notochord); this is the classical developmental example of induction. The cytoskeletal processes underlying neurulation are similar to those underlying gastrulation: cell elongation promoted by microtubules, apical constriction promoted by microfilaments, and convergent extension. The dorsalizing and axis-forming activities of the organizer are due to a program of gene activity initiated by a group of transcription factors expressed in the Spemann organizer. These transcription factors cause expression of a group of genes encoding secreted proteins; these secreted proteins, by antagonizing secreted ventralizing proteins (e. g., BMP4) "induce" the
neural plate and dorsalize the adjacent tissues.
4. NEURAL CREST CELLS
After neurulation, the neural crest cells (NCC’s) leave their position dorsal to the neural tube and migrate throughout the body, giving rise to various tissue types. Some cells take a dorsolateral migration route: they enter the ectoderm (skin) and develop into melanocytes. The melanocytes produce the pigment melanin and are responsible for all of the pigment production in the body (except for the pigment in the pigmented retina of the eye).
Others NCC’s migrate more ventrally and take on a variety of different fates. Some NCC’s migrate just adjacent to the spinal cord; these form the dorsal root ganglia. Others migrate further and form the autonomic nervous system, which consists of the sympathetic ganglia (near the spinal cord) and the parasympthetic ganglia (closer to the target organ). In addition to giving rise to all of the above types of cells in the head, the NCC’s in the head give rise to head mesenchyme, which in turn gives rise to bones, and connective tissue, and the dental papillae (mesenchyme of the developing tooth). Additionally, NCC’s give rise to Schwann cells (which myelinate nerve) and the adrenal medulla (which produces stress hormones).
Transplantation experiments, in which NCC’s from one position in the body are transplanted to another position in the body, indicate that NCC’s are a pluripotent group of cells: they give rise to the cell types expected from the position to which they have been transplanted. Thus, all NCC explants can give rise to parasympathetic ganglia, even though, in situ, parasympathetic ganglia normally only arise from NCC’s of certain locations.
Many different types of experiments have shown that extracellular matrix components (ECM) are critical for the migration of NCC’s.
Analysis of the white mutant in the axolotl (salamander) indicates that ECM is required for NCC migration. In these mutants, NCC’s are present, but they do not migrate beyond their initial position around the spinal cord. The NCC’s themselves seem to be normal, since when transplanted into wild type embryos, they migrate normally. When wild type NCC’s are transplanted into mutant embryos, they do not migrate. That the defect is due to ECM in the mutants was shown by preparing nitrocellulose membranes that had adsorbed ECM in the region where NCC’s would normally migrate. Membranes carrying ECM from normal embryos was able to stimulate NCC migration in both wild type and mutant embryos; membranes carrying ECM from mutant embryos could not stimulate NCC migration, either in wild type, or in mutant embryos.
Experiments in which the function of ECM components are blocked with specific antibodies indicates that ECM components that we have discussed earlier are required for NCC migration. Thus, injection into chick embryos of antibodies against fibronectin, laminin and the fibronectin receptor integrin disturbs the migration of NCC’s. Perhaps not surprisingly, some of the same molecules required for involution of cells during gastrulation (i.e. fibronectin) are required for migration of NCC’s.
Role of dorsal-ventral patterning system (DPP/Sog vs BMP/chordin) in neurulation
In Drosophila, the dorsal-ventral patterning system causes a ventro-lateral portion of the blastoderm layer to become committed to a neurectodermal fate.
The presumptive neurectodermal cells develop from nuclei that contain moderate levels of nuclear dorsal protein. Following cellularization of the blastoderm and the onset of zygotic gene expression, these cells express Sog, an inhibitor of DPP. The neurectoderm in the Drosophila embryo consists of two lateral stripes on either side of the embryo.
These stripes are separated ventrally by the presumptive mesoderm (twist-expressing cells). Following invagination and internalization of the mesoderm, the presumptive neurectoderm comes to occupy the most ventral position in the embryo.
Experiments showing the roles of BMPs and their antagonists in neurulation
Isolated ectodermal cells become neural. However, entire ectodermal explants do not. This is because BMP4 is produced in the ectoderm. As in the mesoderm, BMP is a ventralizing signal. Ventral ectoderm becomes epidermis. Hence, BMP produced by the ectodermal explants induces the entire explant into epidermis.
In the embryo, BMP antagonists secreted by the organizer (dorsal mesoderm) to overlying ectoderm inhibit the ventral fate. Furthermore, Isolated ectodermal cells produce BMP4, but it diffuses away and the ventral fate is thus not induced. Unless overridden by BMP4, ectodermal cells by default differentiate as neural even in the absence of chordin and noggin.
Injecting morpholinos against BMPs into frog embryos will cause excess neural tissue to form. This reinforces the theory that BMPs induce ectodermal cells to become epidermal, and that BMP antagonists prevent this to allow cells to default to a neural state.
Ectoderm appears to undergo a neural path by default, but in fact they have already received neural signals. For example, treatment of embryos with FGFs (fibroblast growth factors) stimulates neural development, and FGFs are produced throughout the embryo.
Neurulation in Drosophila
In invertebrates, neural precursor cells segregate from a specialized region within the ectoderm, called neurectoderm.
The neurectoderm contains cells that will give rise to epidermis (the outer covering of the insect) and cells that will give rise to the nervous system. The neural precursors delaminate individually from the neurectoderm into the interior of the embryo, where they begin to proliferate and are referred to as neuroblasts.
This delamination involves a number of the cytoskeletal and cell adhesion mechanisms that we discussed previously. The delaminating cells lose their connections, via tight junctions and adherens junctions, to cells of the ectodermal epithelium; this is an epithelial to mesenchymal transition. In addition, the delaminating cells constrict at their apical surfaces via microfilaments, and elongate via microtubules.
What is lateral inhibition?
Neuroblasts in insects are determined in a two-step mechanism, the proneural and then the inhibition step.
|Proneural||Proneural clusters are groups of neuroectoderm that become competent to give rise to neuroblasts. This is due to their activation of proneural gene expression. Proneural genes a group of bHLH (basic helix-loop-helix) transcription factors encoded by the Achaete-scute complex. The requirement for the proneural genes is shown by the fact that many neuroblasts do not appear in Achaete-scute-/- embryos.|
|Inhibition||One of the cells that has delaminated from the proneural cluster inhibits other cells of the cluster from becoming neuroblasts. This process is called lateral inhibition, and it is important for assuring that the correct number of cells become neuroblasts.|
Lateral inhibition is mediated by Notch signal transduction. Notch is a transmembrane receptor that interacts specifically with Delta, a transmembrane protein. Therefore, Notch-Delta interaction requires direct cell-cell contact. Initially, all cells in the proneural cluster express low levels of Notch and Delta. One cell in each proneural cluster gains a slight advantage over the others by expressing slightly more Delta. This is likely stochastic (random). This cell thus engages more Notch receptors on neighboring cells, causing them to downregulate proneural gene and Delta expression whilst retaining Notch expression.
These cells thus lose the ability to differentiate as neurons (due to decreased proneural gene expression) and also lose the ability to inhibit proneural expression in other cells (due to decreased expression of Delta). As a result the single cell that expressed slightly more Delta is the only cell that maintains proneural gene expression.
Genes involved in this inhibitory process are called neurogenic genes.
Their required role in this repression is demonstrated by the fact that, in an embryo homozygous for a null mutation in a neurogenic gene, the entire neurectoderm develops as neuroblasts (since all of the cells in the proneural clusters, which constitute the neuroectoderm, now develop as neuroblasts).
Notch and Delta are extremely important neurogenic genes in Drosophila. The Notch receptor and its various ligands have been found to play multiple roles in cell signaling and cell fate determination not only in Drosophila, but also in vertebrates. Moreover, their role in lateral inhibition is largely conserved in vertebrates.
Mechanism of invagination in neurulation
Recall that in amphibian gastrulation, the mesodermal and endodermal cells must enter the interior of the embryo in order to correctly arrange the three germ layers. This is initiated through an invagination, followed by involution.
The dorsal lip of the blastopore is the site where this process is initiated, and it forms opposite the site of sperm entry on the dorsal side of the embryo. Spemann and Mangold wondered if all cells are undetermined at the beginning of gastrulation. They transplanted the dorsal lip of the blastopore of an early gastrula from a light-colored newt into an ectopic region of a dark-colored newt early gastrula.
Spemann and Mangold observed that the donor tissue (dorsal blastopore lip) invaginated into the interior and a second embryonic axis developed. The notochord of this second embryonic axis was composed entirely of graft donor tissue, while the neural tube, and somites (a mesodermal tissue that we will discuss in a later lecture) were composed only partly of graft (donor) tissue. The notochord is the most dorsal mesodermal derivative. All other tissues of the new axis (eg, kidney tubules and gut) were entirely of host tissue. Spemann and Mangold concluded that the graft tissue induced a new embryonic axis.
Noggin, Chordin, Follistatin and Frzb Antagonize BMPs and Wnts
The organizer (induced by the Nieuwkoop Center) dorsalizes the adjacent mesoderm by secreting Noggin, Chordin, Follistatin and Frzb.
These secreted proteins induce the neural plate by antagonizing the ventralizing proteins BMP-4 (noggin, follistatin, and chordin) and Wnt-8 (frzb). Recall that the Organizer organizes the mesoderm around it into an axis by dorsalizing the mesoderm so that dorsal derivative like somites (future trunk muscle and bones) and intermediate derivatives (kidney and gonad) can form. In vertebrates, the most dorsal mesoderm forms the notochord; this requires the dorsalizing Organizer secretions.
Genes encoding Noggin, Chordin, Follistatin and Frzb are able to induce a second axis. Similarly, the major component of the Nieuwkoop center (β-catenin) can also induce a second axis. Thus, the major role of the Nieuwkoop center is to induce the organizer, which in turn produces BMP and Wnt antagonists. For example, injection of mRNA of noggin or chordin into an early embryo can induce a second axis. This second axis is due to the resultant protein's ability to dorsalize ventral mesoderm (resulting in the notochord and somites), and to dosalize the overlying ectoderm (resulting in the neural plate).