NEURULATION

Primary neurulation begins at stage 9 and is completed during stage 12 (Fig. 24.1). The process, although continuous spatially and temporally, has been envisaged as four stages. It begins with local elongation of the ectoderm cells in a midline zone of the embryonic disc and their reorganization into a pseudostratified epithelium, the neural plate. This is followed by reshaping and bending of the neural plate into a neural groove which subsequently closes to form into a neural tube bidirectionally from the midportion to its cranial and caudal ends. A continuous surface ectoderm forms dorsal to the tube.

Fig. 24.1 Scanning electron micrograph of a neurulating rat embryo comparable to a stage-10 human embryo (22–24 days). The expanded brain region can be seen at the top of the image with somite formation occurring centrally as neurulation proceeds caudally. Caudally the ridge of underlying neural crest cells can be seen medial to the unsegmented paraxial mesenchyme.

(Photograph by P Collins; printed by S Cox, Electron Microscopy Unit, Southampton General Hospital.)

Primary neurulation depends on cellular changes within the neural plate: in the trunk it may also involve movements of the paraxial mesenchyme. There is fusion initially of the surface ectoderm followed by fusion of the neural ectoderm. The neural ectodermal cells become elongated and then wedge-shaped. It has been suggested that the forces needed to shape the neural tube are intrinsic to the cells of the neurectoderm. When the neural tube is closing, its walls consist of a single layer of columnar neural epithelial cells, whose extremities abut on internal and external limiting membranes. The columnar cells increase in length and develop numerous longitudinally disposed microtubules. The borders of their luminal ends are firmly attached to adjacent cells by junctional complexes, and the cytoplasmic aspect of the complexes are associated with a dense paraluminal web of microfilaments. The nuclei assume basal positions which, together with the disposition of organelles, impart a slight wedge conformation on some of the cells, and create a hinge point.

The position of hinge points within the neural plate confers different characteristics on the formed neural tube. With a median hinge point, the neural folds remain relatively straight and the tube in this position has a slit-shaped lumen: this can be seen from the initial region of fusion rostralward. If dorsolateral hinge points are added, the resulting neural tube is rhombic, and the hinge points describe the position of the sulcus limitans. If all the neuroepithelial cells exhibit some apical narrowing, then the resulting tube has a circular lumen. The rostral slit-shaped profile of the neural tube may depend more on support from adjacent tissues than the caudal end of the tube, where neurulation is generated by the neuroepithelium. The transition from primary to secondary neurulation (see below) continues the production of a neural tube with a circular lumen.

Fusion of the neural tube starts in embryos with 4–6 somites, at the level of somites 1 and 2, forming the future rhombencephalon. The tube closes caudally and rostrally, forming sequentially cervical and thoracic cord regions, then mesencephalic and prosencephalic brain regions. Rostrally two sites of fusion can be seen. The initial fusion termed α, or the dorsal lip of the rostral neuropore, proceeds caudorostrally. A second site, termed β, or the terminal lip of the rostral neuropore, closes from the rostral end of the neural plate and proceeds rostrocaudally (O’Rahilly & Müller 2002). Closure of these lips of the rostral neuropore is completed when 19–20 pairs of somites are present. Caudal neuropore closure starts when approximately 29 somites are present and the site of closure corresponds to the level of the future somite 31 (the level of the future second sacral vertebra) (O’Rahilly & Müller 2004).

Secondary neurulation starts from the closure of the caudal neuropore at stage 12 and ends at about stage 17; it is the process by which the caudal portion of the neural tube is formed in the absence of a neural plate. At the time of caudal neuropore closure, the caudal midline cells are generically termed the caudal eminence. A specific population of mesenchymal cells, the caudoneural hinge or junction, shares the same molecular markers as the primitive node. These cells aggregate at the midline and undergo mesenchymal/epithelial transformation, producing a cellular cylinder that is contiguous with the caudal end of the neural tube. Further elongation of the caudal neural tube involves cavitation of the neural cylinder. Neural crest cells delaminate from the dorsal surface of the cylinder in a rostrocaudal direction and concurrently, the paraxial mesenchyme undergoes somitogenesis.

EARLY BRAIN REGIONS

Prior to the closure of the neural tube, the neural folds become considerably expanded in the head region; the first indication of a brain and its major divisions can be seen at stage 10 (Fig. 24.2). Regional expansions, prosencephalon (forebrain), mesencephalon (midbrain), and rhombencephalon (hindbrain), have been called the three primary cerebral vesicles, although the term ‘vesicle’ in this context has been considered inappropriate to describe localized accelerations of growth in the wall of the brain. As the neural tube closes, the neural wall appears to form a series of ridges and depressions perpendicular to its long axis. These transient repeating segments are termed primary neuromeres, and initially six can be identified: the prosencephalon, mesencephalon and four subdivisions of the rhombencephalon, rhombomeres A, B, C and D. The primary neuromeres themselves become subdivided during stages 10, 11 and 12 and a total of 16 secondary neuromeres have been identified. The prosencephalon gives rise to the telencephalon, diencephalon 1 (D1) and diencephalon 2 (D2), which has three subdivisions. The mesencephalon is subdivided into mesencephalon 1 (M1) and mesencephalon 2 (M2). The rhombencephalon is subdivided into the isthmus rhombencephali and rhombomeres 1–8; the original rhombomere A gives rise to secondary rhombomeres 1, 2 and 3; rhombomere B gives rise to secondary rhombomere 4; rhombomere C gives rise to secondary rhombomeres 5, 6 and 7; and rhombomere D gives rise to secondary rhombomere 8 (O’Rahilly & Müller 1999). As the rhombencephalon grows the rhombomeric boundaries become less distinct. With the early appearance of the cerebellum from the isthmus rhombencephali and rhombomere 1, the rhombocephalon is traditionally divided into the metencephalon, which extends to about rhombomere 3, and the myelencephalon, which extends to the spinomedullary junction. A summary of the derivative of the cerebral regions is given in Table 24.1 on page 376.

Fig. 24.2 The primary brain regions, neuromeres, and their expansion during neurulation.

Table 24.1 Derivatives of the cerebral regions from caudal to rostral

Rhombencephalon (or hindbrain)
1. Myelencephalon	Medulla oblongata Caudal part of the 4th ventricle Inferior cerebellar peduncles

2. Metencephalon

Pons

Cerebellum

Middle part of the 4th ventricle

Middle cerebellar peduncles

The elongation of the brain occurs at the same time as the appearance of three flexures, which also appear prior to the closure of the neural tube; two are concave ventrally and one concave dorsally. During stages 13 and 14 the brain bends at the mesencephalon (mesencephalic flexure) so that the prosencephalon bends in a ventral direction around the cephalic end of the notochord and foregut until its floor lies almost parallel with that of the rhombencephalon (Fig. 24.3). A bend also appears at the junction of the rhombencephalon and spinal cord (cervical flexure). This increases from the fifth to the end of the seventh week, by which time the rhombencephalon forms nearly a right angle to the spinal cord. However, after the seventh week, extension of the head takes place and the cervical flexure diminishes and eventually disappears. The third bend, the pontine flexure, is directed ventrally between the metencephalon and myelencephalon. It does not substantially affect the outline of the head. In this region, the roof plate thins until it is composed only of a single layer of cells and pia mater, the tela choroidea. The flexure of the neural tube at this point produces a rhombic shape in the roof which later forms the medullary velum.

Fig. 24.3 Developing human brains indicating formation of the mesencephalic, cervical and pontine flexures, lateral views. A, Human embryo, 9 mm long. B, Human embryo, approximately 10.2 mm long. C, Human embryo, 13.6 mm long. The roof of the rhombencephalon has been removed in each case.

EARLY CELLULAR ARRANGEMENT OF THE NEURAL TUBE

Histologically the early neural tube is composed of a pseudostratified neuroepithelium. It extends from the inner aspect of the tube to the outer limiting basal lamina and the surrounding pia mater. The epithelium contains stem cells which will give rise to populations of neuroblasts and glioblasts. A population of radial glia differentiates very early and provides a scaffold for later cells to follow. As development proceeds, three zones or layers develop (Figs 24.4–24.6). These are an internal ventricular zone (variously termed the germinal, primitive ependymal or matrix layer), in which mitosis occurs, and which contains the nucleated parts of the columnar cells and rounded cells undergoing mitosis; a middle, mantle zone (also termed the intermediate zone) which contains the migrant cells from the divisions occurring in the ventricular zone; and an outer, marginal zone, which initially consists of the external cytoplasmic processes of the radial glia. The latter is soon invaded by tracts of axonal processes which grow from neuroblasts developing in the mantle zone, together with varieties of non-neuronal cells (glial cells and later vascular endothelium and perivascular mesenchyme). For further development of these layers see pages 368–369.

Fig. 24.4 Transverse section through the developing spinal cord of a 4-week-old human embryo.

Fig. 24.5 Early development of the neural tube: three layers can be delineated in the spinal cord and brain stem.

Fig. 24.6 The developing spinal cord in the cervical region of a human embryo early in the sixth week; CR length 8 mm. Transverse section.

At first the neural tube caudal to the brain is oval in transverse section and its lumen is narrow and slit-like (Fig. 24.4). The original floor plate and the dorsal site of fusion of the tube initially contain non-neural cells. With cellular proliferation, the lateral walls thicken and the lumen, now the central canal, widens in its dorsal part and is somewhat diamond-shaped on cross-section (Fig. 24.6). The widening of the canal is associated with the development of a longitudinal sulcus limitans on each side which divides the ventricular and mantle (intermediate) zones in each lateral wall into a ventrolateral lamina or basal plate and a dorsolateral lamina or alar plate respectively. This separation underlies a fundamental functional difference.

Throughout the neural tube there is a generic pattern in the position of the neurones which is specified by the juxtaposition of the notochord to the neural tube. Experimental lateral or dorsal grafting of a notochord results in the induction of a floor plate overlying the grafted notochord and the induction of ectopic motor neurones dorsally. Similarly, lateral or dorsal grafts of a floor plate also result in the induction of a new floor plate overlying the graft and the induction of ectopic dorsal motor neurones. Removal of the notochord results in the elimination of the floor plate and the motor neurones and the differentiation of dorsal cell types in the ventral region of the cord (Fig. 24.7).

Fig. 24.7 A–B, Successive stages in the development of the neural tube and spinal cord. A, The neural plate consists of epithelial cells. Cells in the midline of the neural plate are contacted directly by the notochord. More lateral regions of the neural plate overlie the paraxial mesenchyme (not shown). B, During neurulation, the neural plate bends at its midline and this elevates the lateral edges of the plate as the neural folds. Contact between the midline of the neural plate and the notochord is maintained at this stage. C, The neural tube is formed when the dorsal tips of the neural folds fuse. Cells in the region of fusion form the roof plate which is a specialized group of dorsal midline cells. D, Cells at the ventral midline of the neural tube retain proximity to the notochord and differentiate into the floor plate. After neural tube closure neuroepithelial cells continue to proliferate and eventually differentiate into defined classes of neurones at different dorsoventral positions within the spinal cord. For example, sensory relay, commissural and other classes of dorsal neurones differentiate near to the roof plate, and motor neurones differentiate ventrally near the floor plate, which by this time is no longer in contact with the notochord. E–H, A summary of the results obtained from experiments in chick embryos in which a notochord or floor plate is grafted to the dorsal midline of the neural tube or where the notochord is removed before neural tube closure. E, The normal condition, showing the ventral location of motor neurones and the dorsal location of sensory relay neurones. F, Dorsal grafts of a notochord result in the induction of a floor plate in the dorsal midline and ectopic dorsal motor neurones. G, Dorsal grafts of a floor plate induce a new floor plate in the dorsal midline and ectopic dorsal motor neurones. H, Removal of the notochord results in the elimination of the floor plate and the motor neurones and the expression of dorsal cells types in the ventral region of the spinal cord.

The basal plate is normally concerned predominantly with motor function, and contains the cell bodies of motor neurones of the future anterior and lateral grey columns. The alar plate receives sensory inflow from external dorsal root ganglia. Motor and sensory axons combine to form the mixed nerves.

FAILURE OF NEURULATION

Failure of neurulation produces the conditions of craniorachischisis totalis (where the entire neural tube is unfused in the dorsal midline), cranioschisis or anencephaly (where the neural tube is fused dorsally to form the spinal cord but is not fused dorsally in the brain), and spina bifida (where local regions of the spinal neural tube are unfused, or there is failure of formation of the vertebral neural arches). (Fig. 24.8; see also Ch. 44). Anencephalic fetuses display severe disturbances in the shape, position and ossification of the basichondrocranium and in the course of the intracranial notochord and so the condition is more complicated than ‘simply’ a failure of neural tube fusion.

Fig. 24.8 Defects caused by failure of neural tube formation. A, Total failure of neurulation. B, Failure of rostral neurulation. C, Failure of caudal neurulation.

NEURAL CREST

The neuronal populations of the early epiblast become arranged in the medial region of the embryonic disc as the neural plate. Laterally, neural folds or crests indicate the transitional region between neural and surface ectoderm. Along most of the neuraxis the cells at the tips of the neural folds undergo an epithelial/mesenchyme transformation. They acquire migratory properties and leave the epithelium just prior to its fusion with the contralateral fold in the dorsal midline. The migratory cells so formed are collectively termed the neural crest. Cells within the rostral prosencephalic neural fold and smaller populations of cells in bilateral sites lateral to the early brain do not form migratory neural crest cells but remain within the surface epithelium as ectodermal placodes.

Neural crest populations arise from the neural folds as primary neurulation proceeds and simultaneously progresses rostrally and caudally. Crest cells migrate from the neural folds of the brain prior to tube closure. Caudally, from approximately somite 29, secondary neurulation processes produce the most caudal neural crest. Two distinct populations of neural crest cells are formed: a neuronal population produced throughout the brain and spinal cord which gives rise to sensory and autonomic neurones and glia, and a non-neuronal mesenchymal population which arises only from the brain (Figs 24.9 and 24.10). Melanocytes develop from a subpopulation of neural crest cells derived from both the head and trunk. They form one of the three pigment cell types (the others being retinal pigment epithelium and the pigment cells of the pineal organ, which both originate from the diencephalon).

Fig. 24.9 A, Fate map along the neural crest of the presumptive territories which yield the ectomesenchyme, the sensory, parasympathetic and sympathetic ganglia and neural crest derived mesenchyme in normal development. B, Developmental potentials for the same cell types. If neural crest cells from any level of the neural axis are implanted into the appropriate sites of a host embryo, they can give rise to almost all the cell types forming the various kinds of PNS ganglia. This is not true for the neural crest derived mesenchyme whose precursors are confined to the cephalic area of the crest down to the level of somite 5. S, somite.

Fig. 24.10 Fate map of the rostral region of the neural primordium as established by the quail–chick chimera system. A, The various territories yielding rostral head are indicated on the neural plate and neural fold of a 1–3 somite embryo. B, The results obtained in the avian embryo have been extrapolated to the human head. Thus, the neural fold area coloured green in A yields the epithelium of the nose, philtrum and primary palate.

In the trunk the migration patterns of neural crest cells is channelled by the somites. As the crest cells move laterally and ventrally they can pass between the somites and within the rostral sclerotomal half of each somite, but they cannot penetrate the caudal moiety of the sclerotomal mesenchyme. Thus the segmental distribution of the spinal and sympathetic ganglia is imposed on the neural crest cells by a prepattern that exists within the somitic paraxial mesenchyme (Fig. 24.11). The origin of the cranial-caudal patterning of the ventral neural crest cells is not clear.

Fig. 24.11 The migration routes taken by neural crest cells in the trunk.

Rostral to the otic vesicle, neural crest cells arise from specific regions of the brain. Early in development, a number of transverse subdivisions perpendicular to the long axis of the brain can be seen within the rhombencephalon, dividing it into segments termed rhombomeres (Müller & O’Rahilly 1997). Eight main rhombomeres extend from the midbrain–hindbrain boundary rostrally to the spinal cord caudally (Fig. 24.2). Rhombomeres 8 and 7 give rise to neural crest cells which migrate into the fourth and sixth pharyngeal arches; rhombomere 6 crest cells invade pharyngeal arch three. Rhombomere 4 crest cells migrate into arch 2, whereas rhombomeres 5 and 3 give rise to a very small number of neural crest cells which migrate rostrally and caudally to enter the adjacent even-numbered neighbours. Rhombomeres 1 and 2 produce crest cells which invade the first pharyngeal arch. In each rhombomere, mesenchymal populations and the sensory and autonomic ganglia are formed from the crest cells (see Fig. 12.4).

Further rostrally, neural crest from the mesencephalon migrates into the first arch maxillary and mandibular processes. Crest cells are produced from the diencephalon up to the level of the epiphysis. Neural crest cells which are produced from this rostral portion of the brain contribute mesenchymal populations to the frontonasal process. The most rostral prosencephalic neural fold does not give rise to neural crest.

ECTODERMAL PLACODES

Prior to neural tube closure, the elevating neural folds contain two distinctive neuronal populations. The larger population of neural crest cells migrates from the neural epithelium prior to neural tube fusion. A smaller population of neuroepithelial cells becomes incorporated into the surface ectoderm after neural tube closure. These areas of neuroepithelium within the surface ectoderm have been termed ectodermal placodes. Although the majority of the ectodermal placodes form nervous tissue, non-neurogenic placodes also occur (Begbie & Graham 2001). After an appropriate inductive stimulus, the placodes thicken and either generate migratory neuronal cells that will contribute to the cranial sensory ganglia, or the whole placodal region invaginates to form a vesicle beneath the remaining surface ectoderm. Neurogenic placodes undergo both processes. Paired non-neurogenic placodes invaginate to form the lens vesicles under the inductive influence of the optic vesicles (see Ch. 41).

The neural folds meet in the rostral midline adjacent to the buccopharyngeal membrane. This rostral neural fold does not generate neural crest but gives rise to the hypophysial placode, i.e. the future Rathke’s pouch, which remains within the surface ectoderm directly rostral to the buccopharyngeal membrane. The rostral neural fold also gives rise to the olfactory placodes, which remain as paired, laterally placed placodes, and to the epithelium of the nasal cavity (Fig. 24.10).

Further caudally, similar neurogenic placodes can be identified and divided into three categories, namely the epibranchial, otic and trigeminal placodes (Fig. 24.12). The epibranchial placodes appear in the surface ectoderm immediately dorsal to the area of pharyngeal (branchial) cleft formation. The first epibranchial placode is located at the level of the first pharyngeal groove and contributes cells to the distal (geniculate) ganglion of the facial nerve, the second and third epibranchial placodes contribute cells to the distal ganglia of the glossopharyngeal (petrosal) and vagus (nodose) nerves respectively. These placodes thicken and cells begin to detach from their epithelium soon after the pharyngeal pouches have contacted the overlying ectoderm. Concurrently the neural crest cells reach and move beyond these lateral extensions of the pharynx. Neurones migrate from the epibranchial placodes internally to the sites of ganglion formation, where they show signs of early differentiation into neurones, including the formation of neurites.

Fig. 24.12 Scheme of a human embryo of stage 13 (4 mm, 32 somites), rendered flat to eliminate the cerebral flexures. The cranial sensory ganglia and otocyst, derived from the epibranchial placodes are shown on the left. The levels of origin of the neural crest which contributes to the cranial sensory ganglia and mesenchymal populations in the head on the right is indicated. This arrangement may vary in other species. (Provided by Professor R O’Rahilly and Dr F Müller.)

The otic placodes, located lateral to the myelencephalon, invaginate to form otic vesicles from which the membranous labyrinth of the ear develops. Neurones of the vestibulocochlear nerve ganglia arise from neurones that bud off the ventromedial aspect of the otic cup, after which they can be distinguished in the acoustic and vestibular ganglia (see Ch. 38).

The profundal and maxillomandibular trigeminal placodes, which fuse in man to form a single entity, lie rostral of the otic placodes, alongside the mesencephalic–rhombencephalic junction. Prospective neuroblasts migrate from foci dispersed throughout the surface ectoderm lateral and ventrolateral to the caudal mesencephalon and metencephalon to contribute to the distal portions of the trigeminal ganglia.

PITUITARY GLAND (HYPOPHYSIS CEREBRI)

The hypophysis cerebri consists of the adenohypophysis and the neuro-hypophysis. Prior to neurulation the cell populations which give rise to these two portions of the pituitary gland are found next to each other within the rostral portion of the floor of the neural plate and the contiguous midline neural fold. As neurulation proceeds the future neurohypophysis remains within the floor of the prosencephalon. The cells of the future adenohypophysis are displaced into the surface ectoderm, where they form the hypophysial placode in close apposition and adherent to the overlying prosencephalon.

The most rostral portion of the neural plate, which will form the hypothalamus, is in contact rostrally with the future adenohypophysis in the rostral neural ridge, and caudally with the neurohypophysis, in the floor of the neural plate (Fig. 24.10). After neurulation the cells of the rostral neural ridge remain in the surface ectoderm and form the hypophysial placode which is in close apposition and adherent to the overlying prosencephalon.

Neural crest mesenchyme later moves between the prosencephalon and surface ectoderm except at the region of the placode. Before rupture of the buccopharyngeal membrane, proliferation of the periplacodal mesenchyme means that the placode forms the roof and walls of a saccular depression. This hypophysial recess (pouch of Rathke, Figs 24.13 and 24.14) is the rudiment of the adenohypophysis. It lies immediately ventral to the dorsal border of the buccopharyngeal membrane, extending in front of the rostral tip of the notochord, and retaining contact with the ventral surface of the prosencephalon. It is constricted by continued proliferation of the surrounding mesenchyme to form a closed vesicle, but remains for a time connected to the ectoderm of the stomodeum by a solid cord of cells, which can be traced down the posterior edge of the nasal septum. Masses of epithelial cells form mainly on each side and in the ventral wall of the vesicle, and the development of the adenohypophysis progresses by the ingrowth of a mesenchymal stroma. Differentiation of epithelial cells into stem cells and three differentiating types is said to be apparent during the early months of fetal development. It has been suggested that different types of cells arise in succession, and that they may be derived in differing proportions from different parts of the hypophysial recess. A craniopharyngeal canal, which sometimes runs from the rostral part of the hypophysial fossa of the sphenoid to the exterior of the skull, is often said to mark the original position of the hypophysial recess. Traces of the stomodeal end of the recess are usually present at the junction of the septum of the nose with the palate. Others have claimed that the craniopharyngeal canal itself is a secondary formation caused by the growth of blood vessels, and is quite unconnected with the stalk of the adenohypophysis.

Fig. 24.13 Scanning electron micrograph of the roof of the pharynx showing the invagination of placodal ectoderm to form the adenohypophysis (Rathke’s pouch).

(Photograph by P Collins; printed by S Cox, Electron Microscopy Unit, Southampton General Hospital.)

Fig. 24.14 A, B, Sagittal sections of heads of early embryos showing initial stages in the development of the hypophysis.

A small endodermal diverticulum, named Seessel’s pouch, projects towards the brain from the cranial end of the foregut, immediately caudal to the buccopharyngeal membrane. In some marsupials this pouch forms a part of the hypophysis, but in man it is not well defined and disappears entirely.

Just caudal to, but in contact with, the adenohypophysial recess, a hollow diverticulum elongates towards the stomodeum from the floor of the neural plate just caudal to the hypothalamus (Fig. 24.14B); this region of neural outgrowth is the neurohypophysis. It forms an infundibular sac, the walls of which increase in thickness until the contained cavity is obliterated except at its upper end, where it persists as the infundibular recess of the third ventricle. The neurohypophysis becomes invested by the adenohypophysis, which extends dorsally on each side of it. The adenohypophysis gives off two processes from its ventral wall which grow along the infundibulum and fuse to surround it, coming into relation with the tuber cinereum and forming the tuberal portion of the hypophysis. The original cavity of Rathke’s pouch remains first as a cleft, and later as scattered vesicles, and can be identified readily in sagittal sections through the mature gland. The dorsal wall of Rathke’s pouch remains thin and fuses with the adjoining part of the neurohypophysis as the pars intermedia.

At birth the hypophysis is about one-sixth the weight of the adult gland: it increases in weight to become about one-half the weight of the adult gland at 7 years, and attains adult weight at puberty. Throughout postnatal life the gland appears larger in females, in both size and weight.

NEUROGLIA

Glial cells which support neurones in the CNS and PNS are derived from three lineages, namely, neuroectoderm, neural crest and angioblastic mesenchyme. In the CNS, cells of the proliferating ventricular zone give rise to astrocytes and oligodendrocytes. After the proliferative phase the cells remaining at the ventricular surface differentiate into ependymal cells, which are specialized in many regions of the ventricular system as circumventricular organs. In the PNS, neural crest cells produce Schwann cells and astrocyte-like support cells in the enteric nervous system. Angioblastic mesenchyme gives rise to a variety of blood cell types including circulating monocytes which infiltrate the brain as microglial cells later in development.

The ventricular zone lining the early central canal of the spinal cord and the cavities of the brain gives rise to neurones and glial cells (Figs 24.4 and 24.5). One specialized form of glial cell is the radial glial cell, whose radial processes extend both outwards to form the outer limiting membrane deep to the pia mater, and inwards, to form the inner limiting membrane around the central cavity. The geometry of these cells may provide contact guidance paths for cell migrations, both neuronic and glioblastic. A secondary radial glial scaffold is formed in the late developing cerebellum and dentate gyrus and serves to translocate neurones, formed in secondary germinal centres, to their definitive adult locations. Radial glia eventually lose their connections with both inner and outer limiting membranes, except those persisting in the retina as Müller cells, in the cerebellum as Bergmann glia and in the hypothalamus as tanycytes. They can differentiate into neurones as well as astrocytes. They may partially clothe the somata of neighbouring developing neurones (between presumptive synaptic contacts), or similarly enwrap the intersynaptic surfaces of their neurites. Glial processes may expand around intraneural capillaries as perivascular end-feet. Other glioblasts retain an attachment (or form new expansions) to the pia mater, the innermost stratum of the meninges, as pial end-feet. Glioblasts also line the central canal and cavities of the brain as generalized or specialized ependymal cells, but lose their peripheral attachments. In some situations, as in the anterior median fissure of the spinal cord, ependymal cells retain their attachments to both the inner and outer limiting membranes. Thus, glia function as perineuronal satellites, and provide cellular channels interconnecting extracerebral and intraventricular cerebrospinal fluid, the cerebral vascular bed, the intercellular crevices of the neuropil and the cytoplasm of all neural cell varieties.

Microglia appear in the CNS after it has been penetrated by blood vessels and invade it in large numbers from certain restricted regions, whence they spread in what have picturesquely been called ‘fountains of microglia’, to extend deeply among the nervous elements.

MECHANISMS OF NEURAL DEVELOPMENT

For more than a century the mechanisms that operate during the development of the nervous system have been studied experimentally. While much has been established, answers to many fundamental questions still remain obscure. In recent years, significant advances in our understanding of the development of vertebrates have come from work on amphibian, chicken, mouse and fish embryos, using a combination of genetic, embryological, biochemical and molecular techniques to elucidate the mechanisms that regulate the behaviour of early neural populations.

The CNS has a fundamental structure of layers and cells, which are all derived from a pluripotential neuroepithelium. Developing neurones produce axons which traverse great distances to reach their target organs. Within the CNS, they form myriad connections with other neurons in response to locally secreted cues.

HISTOGENESIS OF THE NEURAL TUBE

The wall of the early neural tube consists of an internal ventricular zone (sometimes termed the germinal matrix) abutting the central lumen. It contains the nucleated parts of the pseudostratified columnar neuroepithelial cells and rounded cells undergoing mitosis. The early ventricular zone also contains a population of radial glial cells whose processes pass from the ventricular surface to the pial surface, thus forming the internal and external glia limitans (glial limiting membrane). As development proceeds the early pseudostratified epithelium proliferates and an outer layer, the marginal zone, devoid of nuclei but containing the external cytoplasmic processes of cells, is delineated. Subsequently a middle, mantle layer (intermediate zone) forms as the newly formed neurones migrate ventriculofugally from the ventricular zone (see Fig. 24.5).

Most CNS cells are produced in the proliferative zone adjacent to the future ventricular system, and in some regions this area is the only actively mitotic zone. According to the monophyletic theory of neurogenesis it is assumed to produce all cell types. The early neural epithelium, including the deeply placed ventricular mitotic zone, consists of a homogeneous population of pluripotent cells whose varying appearances reflect different phases in a proliferative cycle. The ventricular zone is considered to be populated by a single basic type of progenitor cell and to exhibit three phases. The cells show an ‘elevator movement’ as they pass through a complete mitotic cycle, progressively approaching and then receding from the internal limiting membrane (Fig. 24.15). DNA replication occurs while the cells are extended and their nuclei approach the pial surface; they then enter a premitotic resting period while the cells shorten and their nuclei pass back towards the ventricular surface. The cells now become rounded close to the internal limiting membrane and undergo mitosis. They then elongate and their nuclei move towards the outer edge during the postmitotic resting period, after which DNA synthesis commences once more and the cycle is repeated. The cells so formed may then either start another proliferative cycle or migrate outwards (i.e. radially) and differentiate into neurones as they approach and enter the adjacent stratum. This differentiation may be initiated as they pass outwards during the postmitotic resting period. The proliferative cycle continues with the production of clones of neurones and glioblasts. This sequence of events has been called inter-kinetic nuclear migration: it eventually declines. At the last division two postmitotic daughter cells are produced and they differentiate at the ventricular surface into ependyma.

Fig. 24.15 The cell cycle in the ventricular zone of the developing neural tube. The nuclei of the proliferating stem cells show interkinetic migration.

(From Journal of Comparative Neurology 120: 37–42, S Fujita, 1963. Reprinted by permission from Wiley-Liss.)

The progeny of some of these divisions move away from the ventricular zone to form an intermediate zone of neurones. The early spinal cord and much of the brain stem shows only these three main layers, i.e. ventricular, intermediate and marginal zones. However, in the telencephalon the region of cellular proliferation extends deeper than the ventricular zone where the escalator movement of interkinetic migration is seen, and a subventricular zone appears between the ventricular and intermediate layers (Fig. 24.15). Here cells continue to multiply to provide further generations of neurones and glia which subsequently migrate into the intermediate and marginal zones. In some regions of the nervous system (e.g. the cerebellar cortex) some mitotic subventricular stem cells migrate across the entire neural wall to form a subpial population, and establish a new zone of cell division and differentiation. Many cells formed in this site remain subpial in position, but others migrate back towards the ventricle through the developing nervous tissue, and finish their migrations in various definitive sites where they differentiate into neurones or macroglial cells. In the cerebral hemispheres, a zone termed the cortical plate is formed outside the intermediate zone by radially migrating cells from the ventricular zone. The most recently formed cells migrate to the outermost layers of the cortical plate, so that earlier formed and migrating cells become subjacent to those migrating later. In the forebrain there is an additional transient stratum deep to the early cortical plate, the subplate zone.

INDUCTION AND PATTERNING OF THE BRAIN AND SPINAL CORD

The generation of neural tissue involves an inductive signal from the underlying chordamesoderm (notochord), termed the ‘organizer’. The observation by Spemann in 1925 that, in intact amphibian embryos, the presence of an organizer caused ectodermal cells to form nervous tissue, whereas in its absence they formed epidermis, led to the discovery of neural induction. However, experiments performed much later in the century revealed that when ectodermal cells were dissociated they also gave rise to neural tissue. The paradox was resolved by the finding that intact ectodermal tissue is prevented from becoming neural by an inhibitory signal(s) that is diluted out when cells are dissociated. Many lines of evidence now indicate that this inhibitory signal is mediated by members of a family of secreted proteins, the bone morphogenetic proteins (BMP). These molecules are found throughout ectodermal tissue during early development, and their inhibitory effect is antagonized by several neural inducers which are present within the organizer, i.e. noggin, chordin and follistatin. Each of these factors is capable of blocking BMP signalling, in some cases by preventing it from binding to its receptor(s).

The regional pattern of the nervous system is induced before and during neural tube closure. Early concepts about regional patterning envisaged that regionalization within mesenchymal populations which transmit inductive signals to the ectoderm impose a similar mosaic of positional values on the overlying neural plate. For example, transplantation of caudal mesenchyme beneath the neural plate in Amphibia induced spinal cord, whereas rostral mesenchyme induced brain, as assessed by the morphology of the neuroepithelial vesicles. However, later work indicated a more complex scenario in which organizer grafts from early embryos induced mainly head structures, while later grafts induced mainly trunk structures. Subsequent molecular data have tended to support a model in which neural-inducing factors released by the organizer such as noggin, chordin and follistatin, neuralize the ectoderm and promote a mainly rostral neural identity. Later secreted signals then act to caudalize this rostral neural tissue, setting up an entire array of axial values along the neural tube. Candidates for these later, caudalizing, signals have been shown to be retinoic acid, fibroblast growth factors and the WNT secreted proteins, which are present in the paraxial mesenchyme and later in its derivatives, the somites. This combination of signals does not seem to be sufficient to produce the most rostral, forebrain structures. Other secreted proteins resident in the rostralmost part of the earliest ingressing axial populations of endoderm and mesenchyme are also capable of inducing markers of forebrain identity from ectodermal cells (Withington, Beddington & Cooke 2001).

As the neural tube grows and is modified in shape, a number of mechanisms refine the crude rostrocaudal pattern which has been imposed during neurulation. Molecules which diffuse from tissues adjacent to the neural tube such as the somites have patterning influences. The neural tube possesses a number of intrinsic signalling centres, such as the midbrain–hindbrain boundary, which produce diffusible molecules capable of influencing tissue development at a distance. In this way extrinsic and intrinsic factors serve to subdivide the neural tube into a number of fairly large domains, on which local influences can then act. Domains are distinguished by their expression of particular transcription factors, which in many cases have been causally related to the development of particular regions. Examples of such genes are the Hox family which are expressed in the spinal cord and hindbrain, and the Dlx, Emx and Otx families of genes which are expressed in various regions of the forebrain. All of these are developmental control genes which lie high up in the hierarchy, and are capable of initiating cascades of expression of other genes to create a more fine-grained pattern of cellular differentiation. In contrast to the aforementioned secreted molecules, these genes encode proteins which are retained in the cell nucleus, and so can act on DNA to induce or repress further gene expression.

Segmentation in the neural tube

The early neural tube is visibly divided into segments, termed neuromeres, by shallow transverse folds which extend perpendicular to its long axis. Primary neuromeres can be identified at stage 9, and 16 secondary neuromeres are present at stage 14. They are especially noted in the rhombencephalon, where they are termed rhombomeres; they have now been shown to constitute crucial units of pattern formation. Domains of expression of developmental control genes abut rhombomere boundaries; single cell labelling experiments have revealed that cells within rhombomeres form segregated non-mixing populations (Fig. 24.16). The neural crest also shows intrinsic segmentation in the rhombencephalon, and is segregated into streams at its point of origin in the dorsal neural tube. This may represent a mechanism whereby morphogenetic specification of the premigratory neural crest cells is conveyed to the pharyngeal arches (see Fig. 12.4). Although these segmental units lose their morphological prominence with subsequent development, they represent the fundamental ground plan of this part of the neuraxis, creating a series of semi-autonomous units within which local variations in patterning can then develop. The consequences of early segmentation for events later in development, such as the formation of definitive neuronal nuclei within the brain stem, and of peripheral axonal projections remain to be explored.

Fig. 24.16 Hox gene expression domains in the branchiorhombomeric area in the mouse embryo at E9.5. The arrows indicate neural crest cells migrating from the rhombencephalon and midbrain. At the former level they are shaded to indicate the Hox genes they express. The same combination of Hox genes is expressed in the rhombomeres and in the superficial ectoderm of the pharyngeal arches at the corresponding rostrocaudal levels. The four Hox clusters are represented below.

(Modified by permission from the Annual Review of Cell and Developmental Biology, Volume 8, 1992 by Annual Reviews www.annualreviews.org.)

Other brain regions are not segmented in quite the same way as the hindbrain. However, morphological boundaries, domains of cell lineage restriction and of cell mixing, and regions of gene expression that abut sharp boundaries, are found in the diencephalon and telencephalon. It is thus likely that compartmentation of cell groups with some, if not all, the features of rhombomeres plays an important role in the formation of various brain regions.

The significance of intrinsic segmentation in the hindbrain is underlined by the absence of overt segmentation of the adjacent paraxial mesenchyme. There is no firm evidence for intrinsic segmentation in the spinal cord. Instead, segmentation of the neural crest, motor axons, and thus eventually the spinal nerves, is dependent on the segmentation of the neighbouring somites. Both neural crest cell migration and motor axon outgrowth occur through only the rostral and not the caudal sclerotome of each somite, so that dorsal root ganglia form only at intervals. The caudal sclerotome possesses inhibitory properties that deter neural crest cells and motor axons from entering. This illustrates the general principle that the nervous system is closely interlocked, in terms of morphogenesis, with the ‘periphery’, i.e. surrounding non-nervous structures, and each is dependent upon the other for its effective structural and functional maturation.

Genes such as the Hox and Pax gene families, which encode transcription factor proteins, show intriguing expression patterns within the nervous system. Genes of the Hox-b cluster, for example, are expressed throughout the caudal neural tube, and up to discrete limits in the hindbrain that coincide with rhombomere boundaries. The ordering of these genes within a cluster on the chromosome (5′-3′) is the same as the caudal to rostral limits of expression of consecutive genes. This characteristic pattern is surprisingly similar in fish, frogs, birds and mammals. Hox genes play a role in patterning not only of the neural tube but also of much of the head region, consistent with their expression in neural crest cells, and within the pharyngeal arches. Disruption of Hox a-3 gene in mice mimics DiGeorge’s syndrome, a congenital human disorder characterized by the absence (or near absence) of the thymus, parathyroid and thyroid glands, by the hypotrophy of the walls of the arteries derived from the aortic arches, and by subsequent conotruncal cardiac malformations. Some Pax genes are expressed in different dorsoventral domains within the neural tube. Pax-3 is expressed in the alar lamina, including the neural crest, while Pax-6 is expressed in the intermediate plate. The Pax-3 gene has the same chromosomal localization as the mouse mutation Splotch and the affected locus in the human Waardenburg’s syndrome, both of which are characterized by neural crest disturbances with pigmentation disorders and occasional neural tube defects. Both Hox and Pax genes have restricted expression patterns with respect to the rostrocaudal and the dorsoventral axes of the neural tube, consistent with roles in positional specification. (For reviews of the expression patterns of these genes see Krumlauf et al 1993.)

While craniocaudal positional values are probably conferred on the neuroepithelium at the neural plate or early neural tube stage, dorsoventral positional values may become fixed later. The development of the dorsoventral axis is heavily influenced by the presence of the underlying notochord. The notochord induces the ventral midline of the neural tube, the floor plate. This specialized region consists of a strip of non-neural cells with distinctive adhesive and functional properties. Notochord and floor plate together participate in inducing the differentiation of the motor columns. Motor neurone differentiation occurs early, giving some grounds for the idea of a ventral to dorsal wave of differentiation. The notochord/floor plate complex may also be responsible for allotting the values of more dorsal cell types within the tube (Fig. 24.7). For example, the dorsal domain of expression of Pax-3 extends more ventrally in embryos experimentally deprived of notochord and floor plate, while grafting an extra notochord adjacent to the dorsal neural tube leads to a repression of Pax-3 expression.

PERIPHERAL NERVOUS SYSTEM

SOMATIC NERVES

Spinal nerves

Each spinal nerve is connected to the spinal cord by a ventral root and a dorsal root (Fig. 24.17). The fibres of the ventral roots grow out from cell bodies in the anterior and lateral parts of the intermediate zone. These pass through the overlying marginal zone and external limiting membrane. Some enter the myotomes of the somites, and some penetrate the somites, reaching the adjacent somatopleure, and in both sites they ultimately form the α-, β- and γ-efferents. At appropriate levels these are accompanied by the outgrowing axons of preganglionic sympathetic neuroblasts (segments T1–L2), or preganglionic parasympathetic neuroblasts (S2–S4).

Fig. 24.17 The developing spinal cord of human embryos: A, approximately 6 weeks; B, approximately 3 months. Transverse sections.

The fibres of the dorsal roots extend from cell somata in dorsal root ganglia (DRG) into the spinal cord and also extend into the periphery. Neural crest cells are produced continuously along the length of the spinal cord, but gangliogenic cells migrate only into the rostral part of each somitic sclerotome where they condense and proliferate to form a bilateral series of oval-shaped primordial spinal ganglia (dorsal root ganglia) (Fig. 24.11). Negative factors in the caudal sclerotome deter neural crest from entering. The rostral sclerotome has a mitogenic effect on the crest cells that settle within it. From the ventral region of each ganglion a small part separates to form sympathochromaffin cells, while the remainder becomes a definitive spinal ganglion (dorsal root ganglion). The spinal ganglia are arranged symmetrically at the sides of the neural tube and, except in the caudal region, are equal in number to the somites. The cells of the ganglia, like the cells of the intermediate zone of the early neural tube, are glial and neuronal precursors. The glial precursors develop into satellite cells (which become closely applied to the ganglionic nerve cell somata), Schwann cells, and possibly other cells. The neuroblasts, at first round or oval, soon become fusiform, and their extremities gradually elongate into central and peripheral processes. The central processes grow into the neural tube as the fibres of dorsal nerve roots, while the peripheral processes grow ventrolaterally to mingle with the fibres of the ventral root, thus forming a mixed spinal nerve. As development proceeds the original bipolar form of the cells in the spinal ganglia changes and the two processes become approximated until they ultimately arise from a single stem to form a unipolar cell. The bipolar form is retained in the ganglion of the vestibulocochlear nerve.

Cranial nerves

Cranial nerves may contain motor, sensory or both types of fibres. With the exception of the olfactory and optic nerves, the cranial nerves develop in a manner similar in some respects to components of the spinal nerves. The somata of motor neuroblasts originate within the neuroepithelium, while those of sensory neuroblasts are derived from the neural crest with the addition in the head of contributions from ectodermal placodes (Fig. 24.18; see Fig. 12.4).

Fig. 24.18 The brain and cranial nerves of a human embryo, 10.2 mm long. Note the derivation of the ganglia associated with the trigeminal, facial, vestibulocochlear, glossopharyngeal, vagus and spinal accessory nerves.

The motor fibres of the cranial nerves which project to striated muscle are the axons of cells originating in the basal plate of the midbrain and hindbrain. The functional and morphological distinction between the neurones within these various nerves is based on the types of muscle innervated. In the trunk, the motor roots of the spinal nerves all emerge from the spinal cord close to the ventral midline to supply the muscles derived from the somites.

In the head the motor outflow is traditionally segregated into two pathways (Figs 24.3B and 24.18). General somatic efferent neurones exit ventrally in a similar manner to those of the spinal cord. Thus the oculomotor, trochlear, abducens and hypoglossal nerves parallel the organization of the somatic motor neurones in the spinal cord. The second motor component, special branchial efferent, consists of the motor parts of the trigeminal, facial, glossopharyngeal and vagus nerves which supply the pharyngeal (branchial) arches, and the accessory nerve. These nerves all have nerve exit points more dorsally placed than the somatic motor system.

The cranial nerves also contain general visceral efferent neurones (parasympathetic preganglionic) which travel in the oculomotor, facial, glossopharyngeal and vagus nerves and leave the hindbrain via the same exit points as the special branchial efferent fibres. All three categories of motor neurones probably originate from the same region of the basal plate, adjacent to the floor plate. The definitive arrangement of nuclei reflects the differential migration of neuronal somata. It is not known whether all these cell types share a common precursor within the rhombencephalon, however, in the spinal cord somatic motor and preganglionic autonomic neurones are linearly related.

These motor neurone types have been designated according to the types of muscles or structures they innervate. General somatic efferent nerves supply striated muscle derived from the cranial (occipital) somites and prechordal mesenchyme. Myogenic cells from the ventrolateral edge of the epithelial plate of occipital somites give rise to the intrinsic muscles of the tongue, while the prechordal mesenchyme gives rise to the extrinsic ocular muscles. Special branchial efferent nerves supply the striated muscles developing within the pharyngeal (branchial) arches (see Fig. 12.4) which are derived from parachordal mesenchyme between the occipital somites and the prechordal mesenchyme. All the voluntary muscles of the head originate from axial (prechordal) or paraxial mesenchyme which renders the distinction between somatic efferent supply and branchial efferent supply somewhat artificial. However, the obviously special nature of the arch musculature, its patterning by the neural crest cells, its particularly rich innervation for both voluntary and reflex activity, and the different origins from the basal plate of the branchial efferent nerves compared to the somatic efferent nerves, make the retention of a distinction between the two of some value.

General visceral efferent neurones (parasympathetic preganglionic) innervate glands of the head, the sphincter pupillae and ciliary muscles, and the thoracic and abdominal viscera.

The cranial sensory ganglia are derived in part from the neural crest, and in part from cells of the ectodermal placodes (Figs 24.12 and 24.18). Generally, neurones distal to the brain are derived from placodes while proximal ones are derived from the neural crest (Fig. 24.18). Supporting cells of all sensory ganglia arise from the neural crest. The most rostral sensory ganglion, the trigeminal, contains both neural crest and placode-derived neurones that mediate general somatic afferent functions. In the case of more caudal cranial nerves (the facial, glossopharyngeal and vagus), the same applies, but the two cell populations form separate ganglia in the case of each nerve. The proximal series of ganglia is neural crest derived (forming the proximal ganglion of the facial nerve, the superior ganglion of the glossopharyngeal nerve and the jugular ganglion of the vagus) while the distal series is derived from placodal cells (forming the geniculate ganglion of the facial nerve, the petrosal ganglion of the glossopharyngeal nerve and the nodose ganglion of the vagus). These ganglia contain neurones that mediate special, general visceral and somatic afferent functions. The vestibular ganglion contains both crest and placodal cells and the acoustic ganglion contains only placodal neurones: the axons from these cells are special somatic afferents and they all travel in the vestibulocochlear nerve.

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