DEVELOPMENT

 

The nervous system of the vertebrates is a complex and intricate system in charged of detecting changes in the internal and external environments, and to bring about actions. It is also the site of higher functions as memory and consciousness.

The development of the nervous system, neurulation, begins by the end of gastrulation where the three germinal layers become stablished: ectoderm, mesoderm and endoderm. In this lecture we will focus on gastrulation, neurulation, and the development of cephalic and axial structures that protect the central nervous system.

GASTRULATION

Gastrulation begins with the formation of the primitive streak on the surface of the epiblast. Cells from the epiblast move and accumulate on the caudal pole of the blastodisc in order to form the primitive streak that grows cranially. As the epiblast cells proliferate, two ridges are formed on each side of the primitive streak, which appears to sink between them. The lower midline portion of the streak is termed the primitive groove. The cranial tip of the groove is called primitive node or Hensen’s node.

 

Before the primitive groove has reached its maximum length, the epiblastic cells start to move towards the groove. From this time on, the primitive streak is a mas of cells in movement. The first epiblastic cells to enter the primitive groove invade the hypoblast and form the intraembryonic endoderm. The original hypoblastic cells moved away and become the extraembryonic endoderm. This process is responsible for the disappearance of great quantities of epiblastic cells that are replaced by new ones of adjacent areas. During this process, the primitive streak persists.

 

A second wave of migratory epiblastic cells move move towards the groove and occupy the space between the epiblast and the endoderm in order to form the mesoderm. The cells in or close to the Hensen’s node, accumulate between the epiblast and the endoderm in the midline to form the notochord (axial mesoderm). Some mesodermal cells on the lateral aspects of the cranial portion of the primitive groove, migrate rostrally to locate cranially to the Hensen’s node and to become the prechordal plate. This is located between the notochord and the oropharyngeal membrane. Cells from the prechordal plate are important for forebrain induction and also contribute to the formation of extrinsic ocular muscles in birds. The epiblast cells that do not pass through the primitive groove remain instead within the epiblast population, and give rise to the neural and surface ectoderm of the embryo.

 

As gastrulation proceeds in a cranio-caudal direction, the primitive groove regresses. Close to the end of gastrulation, the primitive groove has been reduced almost completely. The cells migrating through the remaining groove, will become the caudal bud responsible of the secondary neurulation. 

 

As the Hensen’s node moves caudally, the cells of the ectoderm located rostrally to the node increase in high and begin the process of neurulation, forming the neural plate. This plate grows caudally as the primitive groove regresses. So the neurulation starts while the gastrulation movements are still in place.

 

In human textbooks, authors describe a series of transformations concerning the axial mesoderm and the underlying endoderm. As the axial mesoderm or notochord progresses caudally, the cells are arranged in a tube like structure with a central notochordal canal. Although this concept is not well stablished in the literature, it seems that the notochordal cells become contiguous with the embryonic endoderm. Then, the notochordal lumen opens to the viteline sac while it communicates also with the allantoid sac. This transitory opening between the amniotic cavity and the secondary yolk sac is called the neurenteric canal. It’s possible significance is to equilibrate pressures between both sacs.

 

Some authors state that the neuroenteric canal never exists neither in chick nor in mouse embryos. However, the persistence of the so called neuroenteric canal may cause a rare congenital lesion described in humans, the neuroenteric cysts. These are extramedullary cysts in the vertebral canal, lined with endodermal epithelium and a mucus ­secreting columnar epithelium, similar to that of the intestinal or respiratory epithelia. In dogs, Ferrand[1]  found cysts with the presence of mucus production cells and some cells with microvilli in favor of an endodermal origin.

 

NEURAL TUBE FORMATION: NEURULATION

 

The three embryonic layers formed during the process of gastrulation are the source of the needed materials for the development of all the systems and organs of the embryo. The first one to develop is the nervous system through a process called neurulation. When neurulation occurs, the embryo is called neurula.

 

The neural tube formation involves two independent processes: an anterior or primary neurulation for most of the central nervous system, and a posterior or secondary neurulation for de caudal and last sacral spinal segments.

 

In chick embryos, there is an overlapping site where primary neurulation occurs dorsally and the second neurulation takes place ventrally within the same neural tube.

Primary neurulation

 

As gastrulation proceeds craneocaudally, so the beginning of neurulation. The ectoderm overlying the notochord and the prechordal plate thickens, and becomes the neural plate. This marks the beginning of the primary neurulation.

 

First, the midline neuroectodermal cells of the neural plate are attracted by the notochord and diminish its height. This leads to the formation of a hinge that gives rise to a midline sulcus. Second, other two elevated hinge regions develop on both sides longitudinally. This is the site where a special type of cells develops, the neural crest cells. They are considered by some authors as a forth germinal layer as they give rise to a great variety of tissues. These cells are located at the level were the neural plate contacts the adjacent ectoderm. At this point, the cells increase in height giving rise to the neural folds. The adjacent ectoderm pushes the neural folds towards the midline, adding an extra force to the folds towards the midline. Third, when both folds fuse at the midline, the neural crest cells, located at the fusion site, migrate vetrolaterally. In birds, they start to migrate after the closure of the neural tube. However, in mammals, the neural crest cells from the head region migrate before the closure, while the folds are at its high. They give rise to facial bones and head connective tissue. The rest of the neural crest cells migrate after the closure of the neural tube like in birds.

 

In chick embryos, the closure of the neural tube starts at the site of the future midbrain, and progresses rostrally and caudally. The rostral opening is named the rostral neuropore, and is the first to close. The closing of the neural tube progresses in a caudal direction, like a zip, until the caudal neuropore is closed.

 

In mouse embryos, the initial closure of the neural tube occurs in the hindbrain/cervical boundary. Then, the closure progresses rostrally and caudally. Two more sites occur at the level of forebrain/midbrain and at the rostral end of the future forebrain. In human embryos, the closure occurs as a discontinuous process. The initial site of closure is at the level of the rhombencephalon. As in mouse embryos, the closure at the rostral end of the future forebrain also takes place. It is not well stablished if the second closure site is located slightly more caudal than in mouse embryos, in the hindbrain, or it does not take place at all.

 

The primary neurulation ends when the caudal neuropore closes. This neuropore is estimated to lie at the level of the future second sacral segment.

Neural tube defects

 

Neural tube defects (myeloschisis) are conditions in which the neural tube fails to close. Among them, anencephaly is the condition when the rostral neural tube fails to close. This is a neural tube defect in which there is no formation of higher brain centers as the cerebral hemispheres. This malformation is accompanied by the absence of the cranial vault (exencephaly). Other pathological presentations include microcephaly and macrocephaly.

When the neural folds fail to meet at the midline, the sclerotomes are unable to surround the neural tissue and this results in a dorsally opened vertebra wit myeloschisis. The term spina bifida or rachischisis should be applied to a defect in the closure of the vertebral arch with or without defects of the neural tuve (Langman’s Medical Embryology).

 

An spina bifida with meningocele or myelomeningocele is when the meninges or the meninges with neural tissue are located outside of the vertebral canal respectively.

Lumbosacral myeloschisis usually arises as a defect in the closure of the posterior neuropore, whereas sacrococcygeal abnormalities are caused by secondary neurulation defects.

 

Other abnormalities of the spinal cord are overdistension of the central canal (syringomyelia), longitudinal duplication (diplomyelia), and splitting (diastematomyelia). These defects may be associated with rachischisis.

 

An incomplete separation of the neural tube from the surface ectoderm is the cause of dermoid sinuses with or without a lamina defect. A dermoid sinus is a tubular sac extending from the dorsal midline into the underlying tissues. The dermoid sinuses may remain connected to the spinal cord or terminate in a "blind sac”.

 

Secondary neurulation

 

Caudal to the 27th somite pair in chick embryos, and posterior to the caudal neuropore, the primitive groove is replaced by the caudal eminence, also named “end bud” or “tail bud”. The tail bud gives rise to the last sacral spinal segment and caudal spinal segments, and to mesodermal derivatives. Is at this site were the secondary neurulation takes place, and is species-specific.

 

The mesodermal cells of the tail bud condensate to form the caudal portion of the notochord and paraxial mesoderm, and a chord of re-epithelized cells located between the newly formed notochord and the surface ectoderm. This chord undergoes a process of cavitation to become the conus medullaris, the filum terminale, and the ventriculus terminalis. In chick embryos, there seems to be an overlapping between the primary and the secondary neural tubes. In mouse embryos, the lumen is formed in the center of the newly formed chord before contacting with the primary neural tube. In human embryos, the cavity of the primary tube extends into the secondary neural chord condensation.

 

Cavitation does not occur if the primary neurulation is not completed normally. However, in chick embryos, this process occurs autonomously because it has been demonstrated that in embryos exhibiting lumbosacral myeloschisis, the secondary neurulation occurred normally. Failure in secondary neurulation results in sacral agenesis, absence of cloaca, and symmelia (fusion of the hind limbs).

ARCHITECTURE OF THE CNS TISSUE

 

The wall of the primitive neural tube is formed by cells that contact both the lumen and the surface of the tube. This wall is called the germinal neuroepithelium. However, all the nuclei are arranged at different heights because they move inside the cytoplasm as the go through the process of cellular division. The mitosis takes place close to the lumen.

 

Due to the fact that neurons are cells no longer capable of dividing, the stem cells divide symmetrically (horizontally) in order to provide a great number of cells. The mother stem cells stay periventriculary to produce more daughter cells that will keep dividing. However, a daughter cell may stop dividing symmetrically and pass to an asymmetric (vertical) division. The result of this asymmetrical form of division is a cell, that will undergo further divisions, and a cell that can becomes a radial glial cell, a neuroblast or a basal progenitor that move to the outermost part of the neural tube. A radial glial cell is restricted to the generation of a single cell type, glioblast, basal progenitor or a neuroblast. The nuclei of the basal progenitor cells nuclei travel back to the basal side of the ventricular zone to undergo mitosis to increase the number of astrocytes, oligodendrocytes and neurons distal to the apical surface.

The firsts daughter cells migrate short distances using a somal translocation process in which the cell body extends towards the outer region and attaches to the pial surface. Then the nucleus of the cell moves through the cytoplasm to the outer level. At the end of the process, the cell detaches from the periventricular zone. As the development proceeds, the distances increase and the neurons require glial radial guides to support their migration. During development, the radial glial cells radially span the entire width of the developing cerebral wall from the ventricular cavity to the pial surface. At the end of neurogensis, the radial glial cells disappear, and transform into astroglial cells and neurons. Once the neuron has reached is definitive position, stablishes contacts with other neurons. Moving through a changing cellular environment has important effects on the differentiation of neuroblasts. This kind of migration is seen in regions where cells are organized in layers as in the cortex.

 

Another type of migration is tangential. The neuroblasts move parallel to the pial surface to remain subpially or they may take deeper positions. This has been observed at the rhombic lips (forming the cerebellar germinal layer), in the midbrain (forming the substantia nigra), in the brain stem (forming the cuneate nucleus, olivary nucleus, pontine nuclei and tegmental reticular nuclei), spinal cord, and telencephalon (where they organize to form the basal nuclei). 

 

Neural progenitor cells are capable of producing a great variety of neuron types. However, as development progresses, there is a restriction in differentiation. Also a process of natural cell death takes place responsible for the disappearance of 50% or more of the neurons within a brain region. This elimination of an excess of neurons and their connections occurs prenatally to allow a fine and complex network in the postnatal period. Nevertheless, there is a process of neurogenesis and migrations throughout adult life in the subventricular zone (SVZ) and in the granular cell layer of the dentate gyrus of the hippocampus. This germinal matrix appears during the latter third of embryonic development and persists into adulthood. The majority of precursors within this SVZ germinal layer are glial precursors and, a small cell lineage, develop as neurons. This cells migrate from the SVZ to distant areas.

 

The dentate gyrus of the hippocampal formation involves progenitor cells away from the SVZ and close to the pial surface. The process of addition of new cells remain active during postnatal stages and becomes the site of hippocampal adult neurogenesis. The progenitor cells a mixture of stem cells and neuroblasts at different stages of differentiation. These cells migrate away from the VZ towards the hippocampal fissure. During the postnatal stages the proliferation of cells in the gyrus dentatus becomes more restricted and is confined to the subgranular zone where neuronal stem cells remain throughout adulthood.

Due to the fact that mature oligodendrocytes cannot migrate, it is crucial to prevent differentiation before they reach their destination. As immature oligodendrocytes migrate, they settle along fibers and they transform themselves in mature oligodendrocytes. In the absence of neurons, oligodendrocytes make a myelin-like membrane. However, in the presence of neurons, the expression of myelin gene increases. The process of myelination follows a caudorostral direction in the brain, and a rostrocaudal in the spinal cord. This myelination process is achieved at 40 to 50 days postnatally in the mouse and continue up to 20 years in certain association areas of the brain in humans.

 

In the adult, the oligodendrocytes and astrocytes inhibit regeneration within the central nervous system. In this sense, the axonal regrowth and regeneration of nerve fibers is limited in contrast of the peripheral nervous system. Experiments have shown that damaged axons within the central nervous system are able to regenerate in the presence of a peripheral nervous transplant (Richarson[2] and David[3]). However, inflammation seems to increase migration of immature oligodendrocites and remyelination (Tourbah[4]).

 

Organization of the spinal cord

 

In the spinal cord, the newly formed cells migrate to stablish a layer around the neural tube called the mantle layer. This layer layer is formed by neurons and glia, and their axons move to a more outer location called marginal layer that will become the white matter. The underlying germinal epithelium remains as the ventricular layer or ependymal layer.

 

The cells of the mantle layer of the dorsal half (alar plate) of the spinal cord are sensory, while the ones on the ventral half (basal plate) are motor. Inside the lumen, the sulcus limitans marks the division between both plates. In the alar and basal plates, the neurons are arranged according to their function: in the alar plate, the dorsal ones are somatic, and the ventral ones are visceral. In the basal plate, the dorsal ones are visceral and the ventral ones are somatic.

 

Organization of the encephalon

 

The encephalon develops from the rostral end of the neural tube. In this region, three dilations, or vesicles, develop: the prosencephalon (the most rostral), the mesencephalon (in the middle), and the rhombencephalon (the most caudal). The rhombencephalon continues caudally as the spinal cord.

 

When the optic vesicles develop on the ventrolateral walls of the prosencephalon, two bilateral expansions of the rostral most portion of the prosencephalic vesicle begin to develop. This marks the division of the prosencephalon into telencephalon and diencephalon. The telencephalon grows to become the cerebral hemispheres following a ventro-rostro-dorso-caudal direction, and the diencephalon forms the anterior most portion of the brain stem.

 

Caudal to the prosencephalon, the mesencephalic vesicle becomes the mesencephalon. The rhombencephalon is divided into a rostral portion, the metencephalon, and a caudal portion, the myelencephalon. The metencephalon gives rise to the pons and cerebellum, and the myelencephalon forms the medulla oblongata. 

 

As the different cephalic vesicles appear, the central nervous system increases in volume and begins to flexure. This is due to the fact that the growth of the dorsal portions of the neural tube is more prominent than the growth of the ventral ones. The first flexure to form is the mesencephalic flexure on the ventral aspect of the tube. A second flexure, called the cervical flexure, forms between the myelencephalon and the spinal cord also ventrally. Finally, due to the growth of the rhombencephalon, a third flexure or pontine flexure forms dorsally at the junction between the metencephalon and myelencephalon.

 

The cerebrum

 

The development of the cerebrum follows two different patterns: vertically in layers, and horizontally in regions functionally related.

 

Neuroblast from the ventricular zone migrate on glial processes to outer localizations. The neuroblasts produced at early stages migrate to the inner most layers of the cortex while the ones produced at latter stages migrate to outer regions through the previously generated glial cells. As the neocortex and isocortex are made of multiple layers, they follow the mechanism of adding new external layers to the ones already formed. The complete development is achieved after birth. The basal nuclei are formed by tangential migration as reported previously.

The brain stem

 

At the level of the brain stem, the cells of the mantle layer form columns following the same functional pattern described in the spinal cord. In the alar plate, the dorsal are somatic and the ventral are visceral. In the basal plate, the dorsal are visceral and the ventral are somatic. As in the spinal cord, the sulcus limitans marks the boundary between the alar and the basal plates. The formed columns of cells in the mantle layer become divided into nuclei. Due to the changes in shape and volume of the neural tube, some nuclei are displaced from its original position.

 

In the hindbrain, eight segments, the rhombomeres, can be identified. They have variable expression patterns of homeobox genes that specify their derivates. The neural crest cells related to the rhombomeres migrate to populate specific pharyngeal arches.

The cerebellum

 

The cerebellum derives from the rostral portion of the rhombencephalon, the metencephalon. The thin roof of the metencephalon is occupied by neuroepithelial cells coming from two lateral expansions called rhombic lips. These expansions grow on the top of the roof of the fourth ventricle and fuse in the midline to form the cerebellar plate. As a result of the deepening of the pontine flexure, the cerebellar plate is compressed in a rostrocaudal direction forming several furrows on the cerebellar plate. In this plate, a small midline, the vermis, and two lateral portions, the cerebellar hemispheres, may soon be distinguished. As the vermis completes the development latter that the hemispheres, it is most likely to be absent or underdeveloped that other parts of the cerebellum.

 

The mantle layer cells of the cerebellar plate migrate towards the surface and give rise to a cortical layer. The migration of newly formed cells follow the “glial monorail” pattern (following glial processes by adherence proteins) to reach their finally location. Cells from the cortical layer descend and form the granular and Golgi cells, giving room on the surface for the molecular layer to develop. At the same time, some mantle cells ascend to form the Purkinje cells. Their dendrites make up a major portion of the molecular layer and most of their axons are directed towards the deep cerebellar nuclei (fastigial, interpositus and dentatus). These nuclei are derived from cells of the mantle cell layer that have not been displaced dorsally.

 

Phylogenetically the cerebellum can be divided in: paleocerebellum or spinocerebellum, neocerebellum or cerebrocerebellum and archicerebellum or vestibulocerebellum. The paleocerebellum is related to the rostral lobe, paraflocculus, paramedian lobule, pyramis and nodulus. The neocerebellum refers to the declive, folium, tuber, simplex lobule and ansiform lobule. The archicerebellum is formed by the floculonodular lobe.

 

Choroid plexuses

 

The choroid plexuses develop on specific locations: on the medial aspect of each developing telencephalic vesicle, and on the roof of the third and fourth ventricles. At these sites, the piamater contacts the ependymal cell layer. The combined tissue is called tela choroidea. As the blood capillaries run in the subarachnoid space in close contact with the piamater, the tela choroidea plus blood capillaries forms the choroid plexuses. These plexuses are located inside the lateral ventricles and extend, through the interventricular foramina, to the third ventricle forming two longitudinal rows on the roof of this ventricle. Two more rows of capillaries develop on the tela choroidea of the fourth ventricle, caudally to the caudal medullary velum.

 

Each choroid plexus projects into the ventricle as a band of clustered villi. They produce cerebrospinal fluid by secretion and ultrafiltration.

ORGANIZATION OF THE PARAXIAL MESODERM

 

Lateral to the neural tube, the paraxial mesoderm condensates along the length of the embryo during gastrulation to form the somitomeres. They appear in pairs, beginning at the head region, and extending caudally. When the nervous system begins to develop, each neuromere is related to a somitomere. The somitomeres located lateral to the developing encephalon contribute to form the cranial muscles, most of the neurocranium, and the dermal and meningeal tissues. In the head, the neural crest contributes to the formation of the splacnocranium together with the mandible and the hyoid bone, the connective tissue of the head muscles, and to the wall of the blood vessels.

 

Caudal to the seventh somitomere, the somitomeres condensate and separate into units to become somites. As the somite matures, three different regions can be identified: dermatome, sclerotome and myotome. The dermatome gives rise to the dermis and subcutaneous tissue. The sclerotome, to the vertebrae and ribs. The miotome forms the axial, thoracic, abdominal and limb striated muscles. The first four/five somites are named occipital somites.

 

The vertebrae derive from the ventromedial part of the somites, known as the sclerotome. The sclerotomes undergo a process or resegmentation to allow the spinal nerves to exit the vertebral canal. Without this process the muscles would originate and terminate in the same vertebra impeding the movement. Due to the increased cell proliferation on the caudal half of each sclerotome, two parts can be distinguished: a loose cranial portion and a dense caudal portion. The latter extends caudally to join the cranial part of the next sclerotome. This process leads to the future vertebra to be located intersegmentally.

 

The ventral part of the sclerotome proliferates surrounding the notochord to become the vertebral body and the ribs. The lateral part of the sclerotome grows dorsally to form the vertebral arch. The anulus fibrosus of the intervertebral disc and cartilage end-plate derive entirely from the sclerotome, and the nucleus pulposus from the notochord. Cells from the cranial loose part of the sclerotome surround the notochord between the vertebral primordia to become the anulus fibrosus and the notochord trapped inside becomes the nucleus pulposus. However, there is controversy over the origin of the sclerotomal cells, being from the loose or from the dense portions. Concerning the nucleus pulposus, two theories coexist. One advocates that regional apoptosis and proliferation of the notochordal cells trapped in the vertebral body lead to notochord removal in the vertebral body, and expansion into the intervertebral disc. Other, supports that pressure exerted by the surrounded vertebral body on the notochord is responsible for pushing the notochord cells into the intervertebral disc region (Sivakamasundari[5]).

 

During the process of resegmentation of the sclerotomes, the neural crest cells move ventrolaterally under the ectoderm, and trough the loose part of the sclerotome (Noden[6]). They form sensory neurons and the spinal ganglia. The ventral root axons move also through the loose part, guided by the neural crest cells. Some neural crest cells continue to migrate towards the aorta to become postganglionar autonomic cells and adrenomedullary cells. The rest move to the primitive intestine.

 

The first four sclerotomes form the occipital and otic regions of the skull. The fourth and fifth sclerotomes form the proatlas. The anterior segment of the proatlas becomes the occipital condyles, and the posterior segment forms the articular surfaces of the atlas and the apex of the dens of the axis. The mesenchyme of the fifth and sixth sclerotomes form the primordial body of the atlas and the dens of the axis. The body of the axis develops from the sixth and seventh sclerotomes. The C3 vertebra is formed from the seventh and eight sclerotomes, and so on. As a consequence, each cervical spinal nerve exits cranially to the vertebrae with the same number. However, as there are 8 cervical spinal nerves and 7 cervical vertebrae, all the thoracic, lumbar, sacral, and caudal nerves exit caudally to their corresponding vertebra.

 

During the early stages of development, the neural tube and the vertebral column develop at a similar rate. However, as the embryo develops, there is a differential growth between the spinal cord and the vertebral column. This leads to a difference in length between the two structures, resulting in a shorter spinal cord. This difference in growth continues till adulthood. In an adult midsize dog, the spinal cord reaches the sixth or seventh lumbar vertebrae.

Vertebral malformations

 

When a homologous sclerotome fails to develop or there is a failure in the condrification or ossification centers of the vertebral body, just one half of the body develops resulting in asymmetry. The wedge-shaped vertebra is called hemivertebra. The shape of this vertebra depends on the area that fails to develop. The most commons are lateral and dorsal hemivertebrae. Scoliosis is often present at birth. The absence of the whole body is termed asomia (agenesis). When the notochord fails to disappear at the body of a vertebra, the resulting shape of the vertebra a butterfly vertebra. A transitional vertebra is a vertebra having features of two types regions. The most common is in the lumbosacral region with a vertebra with features of both lumbar and sacral vertebrae.

 

 

[1] Ferrand, F.X. et al. Spinal neurenteric cyst in a dog. Irish Veterinary Journal  68:9, 2015

[2] Richardson, P.M., MCguiness U.M., and Aguayo, A.J. Axons from CNS neurons regenerate into PNS grafts. Nature 284: 264–265, 1980.

[3] David, S. and Aguayo, A. Axonal elongation into peripheral nervous system “bridges” after central nervous system injury in adult rats. Science 214: 931–933, 1981.

[4] Tourbah, A., linnington, C., Bachelin, C., Avellana-Adalid, V., Wekerle, H., and Baron-Van Evercooren, A. Inflammation promotes survival and migration of the CG4 oligodendrocyte progenitors transplanted in the spinal cord of both inflammatory and demyelinated EAE rats. J Neurosci Res 50: 1–9, 1997.

[5] Sivakamasundari, V. and Lufkin, T. Bridging the Gap: Understanding Embryonic Intervertebral Disc Development. Cell Dev Biol. 1:103. 2012

[6] Noden, D.M.  and Trainor, P.A. Relations and interactions between cranial mesoderm and neural crest populations. J. Anat. (2005) 207, pp575–601