Development of the Nervous System

Introduction

The human brain arises from a restricted population of embryonic cells to become the most complex organ system known during the brief 280 days of human gestation. The newborn brain is comprised of billions of neurons and glia arranged and interconnected in an exquisitely precise three-dimensional network. Unfortunately, minor changes have profound implications for postnatal development and function. Overlapping genetic and epigenetic events during neurodevelopment are tightly regulated in both time and space, transforming a thin disk of undifferentiated neuroepithelium into a complex multilayered system. The brain, with its massive prefrontal cortex and ability to investigate and reflect on its own nature and function, primarily differentiates human fetal development from that of other species. In this chapter we review the major temporal events in prenatal nervous system development together with several common disorders and anomalies that result from perturbations of these pathways. An overview of significant stages of central nervous system (CNS) development and the disorders associated with their interruption in fetal life is provided in Table 124.1 .

Embryogenesis and Early Formation of the Nervous System

The fertilized human ovum divides symmetrically into identical totipotent blastomeres, and within a few cell divisions a blastocyst forms, uterine implantation occurs, and the inner cell mass contains cells committed to formation of the embryo. The progressive specialization of cells is highly dependent on extrinsic factors in the surrounding environment and intrinsic genomic expression. The temporal and spatial sequence of events during early development is tightly regulated. The nervous system is sensitive to genetic mutation (inherited or de novo) and local, epigenetic disruptions that may lead to similar or overlapping anomalies. ^,

One of the first steps in successful brain development occurs in the third week of gestation when the bilaminar inner cell mass, composed of the epiblast and the hypoblast, undergoes gastrulation to form the three embryonic germ layers (endoderm, mesoderm, and ectoderm). The process begins with the appearance of the primitive streak ( Fig. 124.1 ) on the epiblast surface and a defined cephalic pole, the primitive node. Formation of the streak is controlled primarily by the activation of the Wnt pathway. The epiblast gains the ability to migrate towards the primitive streak through the process of epithelial-to-mesenchymal transformation. Epithelial-to-mesenchymal transformation and migration towards the streak occurs under control of the Wnt pathway, the transforming growth factor β family member Nodal, and fibroblast growth factor 8, a growth factor synthesized by streak cells. Zinc finger transcription factors, SNAI1 and SNAI2, are mediated by fibroblast growth factors to down-regulate E-cadherin, the molecule responsible for epiblast binding. Decreased cell adhesion allows migrating epiblast cells to invaginate in the region of the node and streak, and delaminate and displace hypoblast cells to form the endoderm and mesoderm. Remaining epiblast cells differentiate into ectoderm. The dorsal mesoderm gives rise to the notochord. The notochord then induces the overlying ectoderm to thicken and form the neural plate and subsequently gives rise to neuroectoderm, marking the end of gastrulation and the beginning of neurulation.

Gastrulation also marks the visible establishment of primary axes of the embryo and nervous system: lateral, anteroposterior, and dorsoventral. The future complex shape and three-dimensional structure of the fetal brain is determined by patterning events that begin with the establishment of the neural placode and plate. Two sets of polarity are required. The first is anteroposterior regional differentiation into future forebrain, midbrain (“neuromeres”), hindbrain (subsegmented into “rhombomeres”), and spinal cord, due to initial clonal restriction and refinement under the influence of local neural plate “organizers” and anteroposterior regionally restricted regulatory transcription factors. The second is establishment of the dorsoventral polarity that sequestrates motor neurons from sensory neurons in the developing spinal cord, and eventually cerebral cortical regionalization into recognizable functional areas such as motor or visual cortex. Further, positional cues are conferred by mesodermal structures such as notochord and prechordal mesoderm. Intricate timing and precise localization of intracellular and extracellular factors are key in the establishment of the fetal brain.

Three major steps are required in the formation of the prospective forebrain. Ectodermal cells must acquire neural identity, rostral neural tissue must adopt anterior character, and regional patterning must occur within the rostral neural plate. In addition to the multiple feedback and feed-forward loops that exist, many factors are expressed several times throughout development, adding to the complexity of the system.

Neural induction is the process by which embryonic cells in the ectoderm acquire a neural fate (to form the neural plate) rather than giving rise to other structures such as epidermis or mesoderm.

Ectodermal cells will acquire an anterior neural fate without extracellular cues. However, multiple extracellular signaling molecules are involved in the establishment of the rostral-caudal axis. Fibroblast growth factors, Wnts, retinoic acid, Nodals, and bone morphogenetic proteins (BMPs) are among the signaling molecules that have been proposed as caudalizing factors, which inhibit neuronal fate. Localized expression of the caudalizing factors, localized expression of caudalizing factor antagonists in rostral tissue, and morphogenetic movements keeping the anterior neural plate out of the range of the factors are the mechanisms by which the axis is formed.

BMPs and the transforming growth factor β signaling pathway promotes an epithelial fate and are potent inhibitors of neural differentiation. If the BMP signal is blocked by one of many extracellular antagonists derived from adjacent cells, the Smad pathway is inhibited, allowing expression of the Sox proteins that activate transcription of proneural basic helix-loop-helix class genes. These genes code for a class of more than 20 transcription factors necessary and sufficient for nervous system formation, some expressed throughout the developing nervous system, others specific to regions such as the forebrain and spinal cord. Overall, if BMP signaling is too high, nonneural fates are promoted, whereas if it is too low, then more medial neural plate fates are promoted. ^,

Similarly, ectodermal cells acquire a neural identity through expression of antagonists of the canonical Wnt. Antagonism of the Wnt pathway occurs by extracellular proteins, such as the secreted frizzled-related protein, and intracellular factors—for example, the scaffolding protein axin 1 and the transcriptional repressor transcription factor 3. In addition, the transcription factors SIX3 and IRX are expressed in anterior and posterior regions of the neural plate, respectively, and act in a mutually repressive fashion in response to Wnt activity.

The Notch/Delta signaling pathway allows delamination of ectodermal cells to become neuroblasts (i.e., committed neural progenitors) while laterally inhibiting other ectodermal cells from following this fate. As a result of these signals, cells destined to form the neural plate elongate in an apical-basal direction to form the neural placode, broader at its cranial end and narrowing caudally.

Homeobox gene clusters encode transcription factors that establish an anteroposterior axis and control body segmentation through formation of somites. Within the homeobox clusters, forebrain and midbrain are specified by the OTX (orthodenticle homologue) gene family. OTX genes are expressed before the EMX (empty spiracles homologue) genes, which are expressed only in the forebrain. The hindbrain is specified by expression of the earliest HOX genes, HOXA1 , HOXA2 , and HOXB2 . Expression and repression of these patterning genes is controlled primarily by gradients of the multiple extracellular signaling molecules.

Our understanding of early neuronal patterning has been refined due to novel genetic techniques and the development of mammalian embryonic stem cell systems. The number of factors impinging upon the BMP, Wnt, and Notch/Delta signaling pathways has grown. Recent studies have added to the complexity of early patterning that demonstrates epigenetic chromatin-remodeling events that preconfigure epiblast cells to respond to these extracellular cues. The precise interaction across these pathways and the contribution of epigenetic control mechanisms remain to be fully elucidated.

Neurulation and Formation of the Spinal Cord

Neurulation is the process of formation of the hollow neural tube by folding of the epithelial neural plate ( Fig. 124.2 ). Neurulation in humans occurs in two distinct phases: primary neurulation during weeks 3 and 4 of gestation leading to development of the brain and spinal cord ( Fig. 124.3 ), and secondary neurulation during weeks 5 and 6, with formation of the lower sacral and coccygeal cord. Defects of neurulation are the earliest abnormalities of brain development that are clinically detectable in fetal life and extend into postnatal life.

The neural plate elongates into a drop-shaped structure, broader at its cranial end and narrower in the future spinal regions. This morphogenetic event, known as convergent extension, is under the control of the noncanonical Wnt pathway and downstream proteins such as VANGL1, CELSR1, SCRB1, and Dishevelled.

The neural plate is further shaped by bending at the median hinge point overlying the notochord at the future upper spinal cord and at the paired dorsolateral hinge points at the levels of the brain and lower spinal cord. This differential bending appears to be under the control of signals diffusing from the notochord, including the signal transduction molecule sonic hedgehog (SHH), which is also the major determinant of ventral neural progenitor identity in embryonic spinal cord and forebrain. ^, Differential gradients of expression between the ventral SHH expression and BMPs in the dorsal ectoderm not only establish a dorsoventral plane but also lead to the later establishment of distinct classes of neurons with the spinal cord. ^, The neural plate rotates by elevation and convergence around the median and dorsal hinge points. This bending appears to be dependent on apical constriction of columnar neural tube cells to become wedge-shaped under the control of actin-related genes, such as ARHGAP35 (a negative regulator of Rho GTPase), MARCKS (a protein kinase C target), shroom genes (which encode an actin-associated protein family), and VCL (which encodes the actin-binding protein vinculin). Cytoskeletal proteins appear to regulate only cranial neurulation, whereas the spinal neural tube closes normally despite defects in several cytoskeleton-related genes.

Neural tube closure appears to continue by the development of bilateral folds at its junction with nonneural ectoderm. These folds elevate and become opposed in the midline, with fusion occurring by interdigitation of cellular protrusions from apical cells and the formation of permanent cell contacts. Membrane-bound ephrin ligands and their Eph tyrosine kinase receptors have been implicated in this process. Maintenance of proliferation by Notch signaling and apoptotic programmed cell death play important but poorly understood roles in the process as well.

Neural tube closure is a discontinuous process occurring at two invariant sites in humans, with a third variable site representing a potential factor for neural tube defects (NTDs). The open neural folds between initiation closure sites are known as neuropores. As closure progresses, the neuropores gradually shorten and close, leading to an intact closed neural tube. This is subsequently covered by ectoderm-derived epidermis.

Secondary neurulation is initiated after primary neurulation is complete and the posterior neuropore closes. The tail bud, a pluripotent mass of cells, a remnant of the caudal primitive streak, proliferates and condenses, followed by cavitation and fusion with the central canal of the neural tube. As this process of canalization progresses during ensuing weeks, neurons and ependymal cells differentiate to form the caudal end of the spinal cord. Resorption of the tail bud and other cells of the caudal cell mass leaves the filum terminale, which often contains ependymal cell nests along its length. These nests of ependymal cells can begin to proliferate later in life as a monoclonal population of glial cells. These ependymomas, uncommon glial cell tumors, are among the most common tumors of the cauda equina and filum terminale.

The closed neural tube consists of a thick pseudostratified epithelium of neuroepithelial cells that begin to divide rapidly immediately after closure and give rise to a second cell type, the neuroblast. These cells form the mantle layer, the future gray matter of the spinal cord. This further gives rise to an outermost layer, the marginal layer that will become myelinated and form the future white matter of the spinal cord. As neuroblast proliferation continues, the neural tube develops dorsal and ventral thickenings that become the alar and basal plates that will give rise to the sensory and motor areas of the spinal cord, respectively. A longitudinal groove, the sulcus limitans, demarcates the boundary between the two.

As the neural tube closes, cells at the edge of the neural plate separate from the neural epithelium and migrate into the extracellular matrix to become neural crest cells. Neural crest migration is required for complete closure of the cranial neural tube but not for spinal neural tube closure. Although cell adhesion molecules were previously thought to play a role in neurulation, more recent studies have shown mice with null mutations in neural cell adhesion molecule (NCAM) or N-cadherin undergo normal neurulation. Down-regulation of cell adhesion molecules, transition from tight junction to gap junction molecules, and an increased expression of matrix metalloproteinases play an important role in neural crest migration.

Neural crest cells migrate widely to become neurons and glia in dorsal root and autonomic ganglia. The fate of neural crest–derived cells is also to provide innervation for the gastrointestinal tract and to become neurons in the sensory ganglia of the cranial nerves (also formed in part by ectodermal placodes) and to become melanocytes, cartilage of the bone and face, and a variety of endocrine and structural tissues.

The ultimate phenotypic and developmental fate of neural crest cells is tied to the timing, mode, and pattern of migration and is in large part controlled by the environment in which they reside. In addition to various permissive versus inhibitory signaling molecules including Eph/ephrin, semaphorin/neuropilin, and Robo/Slit, a complex relationship exists between neural crest cells and the developing vasculature. The dorsal aorta, the first major blood vessel established during embryogenesis, expresses BMP signals to direct migration and lineage segregation of neural crest cells into adrenal medulla or sympathetic ganglia. Schwann cells, neural crest–derived glia that ensheath peripheral nervous system neurons, direct patterning of arterial vasculature parallel to sensory nerves through the expression of CXCL12 (also known as SDF1 ). Neural crest cells retain a relatively broad developmental potential as they begin migration and their ultimate fate is strongly influenced by local factors.

Neural Tube Defects

NTDs are collectively the most common malformation of the nervous system, with an incidence of 1 to 2 per 1000 births, although there is significant geographic and historic variation in prevalence. This variation hints at the complex cause of these disorders, in which both genetic and environmental factors appear to be significant, including maternal age and diet, maternal diabetes and obesity, teratogen exposure, and socioeconomic class. Pathologically, NTDs are characterized by a failure of closure of the neural tube at any level. Failures of primary neurulation frequently lead to “open” NTDs, in which the neural defect is exposed to the environment, whereas failures of secondary neurulation frequently lead to “closed” defects, in which the defect is covered by skin and integuments. ^, Open NTDs are usually associated with other CNS anomalies, in contrast to closed NTDs, where associated CNS anomalies are rare.

Myelomeningocele

Failure of posterior neural tube closure results in a malformation of the spinal cord, myelomeningocele. This is the most commonly encountered NTD in pediatric practice because most infants born with this anomaly survive. Valproic acid exposure increases the risk 10-fold, and diabetic mothers are at increased risk as well. The lesion consists of neural plate or rudimentary neural tube tissue herniating through a defect in the vertebra (spina bifida) in the form of a sac containing meninges, cerebrospinal fluid (CSF), nerve roots, and spinal cord. The dorsal part of the cord is most affected, and there may be some dermal coverings. The vast majority of myelomeningoceles occur in the lumbar region ( Fig. 124.4B ), and 10% may contain no neural tissue and are known as meningoceles . Spinal cord lesions above the myelomeningocele, such as syringomyelia, hydromyelia, and split cord, are common. Hydrocephalus, Arnold-Chiari malformations, and cortical dysplasias are frequent associations. Fetal closure of a lumbosacral NTD is associated with better long-term outcomes, suggesting that secondary effects of a prolonged open NTD can be assuaged.

Disorders of Neural Crest–Derived Cells

Defects in neural crest development are known as neurocristopathies. Many of the neurocutaneous disorders are a result of defects in neural crest tissue. Tuberous sclerosis and neurofibromatosis will be discussed later in this chapter but are a result of overgrowth of cells originating from the neural crest. In Waardenburg syndrome, children have hypoganglionosis, congenital megacolon, abnormalities of the great vessels, sensorineural deafness, and abnormalities of skin, hair, and iris pigmentation. Congenital aganglionosis (Hirschsprung disease) is characterized by a lack of neurons in the enteric plexus of the colon as a result of either failure of migration or failure of differentiation of neural crest cells. A mutation in L1CAM , a gene that encodes a neural cell adhesion molecule, has been described in newborn males with congenital hydrocephalus, aqueductal stenosis, and/or agenesis of the corpus callosum and Hirschsprung disease, suggesting a role for cell adhesion mediated by L1 cell adhesion molecule.

Disorders of the spinal and autonomic ganglia are rare. Congenital insensitivity to pain with anhidrosis is an autosomal recessive disorder resulting from defective neural crest differentiation. The presence of multiple coincident neurocristopathies has been reported, specifically with the association between congenital central hypoventilation, Hirschsprung disease, and autonomic dysfunction, with PHOX2B the main disease-causing gene identified.

Embryonic Development of the Brain and Ventricular System

The brain forms from three vesicles that develop in the cranial neural tube: the forebrain (prosencephalon), midbrain (mesencephalon), and hindbrain (rhombencephalon). This process occurs at weeks 4 and 6 of gestation in humans shortly after closure of the anterior neuropore. Inductive interactions between the rostral notochord–prechordal mesoderm and the most cranial portion of the neural tube initiate formation of the fetal forebrain. As this process occurs in the ventral surface of the rostral neural tube, it is often known as ventral induction . This induction influences the formation of much of the face and forebrain, thus disruption of this process leads to severe brain disorders and accompanied facial anomalies. Ventral induction is followed by the cleavage of the forebrain in three planes: horizontally, to give rise to the paired optic vesicles, olfactory bulbs, and tracts; transversely, to separate the diencephalon from the telencephalon; and finally, sagittally, to form the paired cerebral hemispheres, lateral ventricles, and basal ganglia.

During the fifth week of gestation the cerebral hemispheres develop as symmetric invaginations of the lateral wall of the prosencephalon. The median portion of the prosencephalon forms the diencephalon, giving rise ultimately to the third ventricle, thalamus, hypothalamus, and mammillary bodies. The cavities of the hemispheres form the lateral ventricles and communicate with the lumen of the diencephalon through the foramina of Monro. The basal parts of the hemispheres begin to grow and bulge into the floor of the foramina to form the ganglionic eminence and ultimately the caudate and lentiform nuclei. Clusters of neurons along the midline begin to form nuclei of the brain stem, thalamus, and hypothalamus. The remarkable expansion of the cerebral hemispheres follows during the remainder of gestation.

Transcription factors, such as FOXG1, GLI3, and PAX6, among others, play significant roles in dividing the telencephalon into dorsal and ventral sections. ^, FOXG1 is expressed in the anterior plate cells and induces the expression of fibroblast growth factor 8 to positively regulate ventral telencephalic development. PAX6 is essential for setting up the sharp border between the dorsal and ventral telencephalon. GLI3 expression is important for dorsal telencephalic development and induction of the Wnt and BMP signaling pathways. BMP signaling appears to be required for formation of the dorsal midline structures through the nodal pathway and induction of transcriptional regulators TGIF, TDGFI, and FASTI. Interaction between BMP and Wnt signaling appears to be important for hippocampal formation. The transcription factors discussed and others, such as COUPTF1, EMX2, and LHX2, are responsible for cortical and subcortical development and regionalization.

The SHH signaling pathway, activated by secretion of SHH proteins from the prechordal mesoderm, is critical to the development of the ventral prosencephalon. SHH is a secreted signaling molecule that undergoes cleavage into amino-terminal and carboxyl-terminal domains. The amino-terminal domain binds to the patched 1 receptor to suppress its repression of the G protein–coupled receptor Smoothened, which activates downstream signaling. A key role for SHH is to antagonize GLI3-mediated repression of ventral telencephalic and spinal cord gene expression and thus promote ventral identities. For example, SHH is critical for thalamic patterning and hypothalamus formation, because all hypothalamic tissue is absent in mice lacking SHH activity. This requirement is observed for most ventral neuronal populations throughout the neuraxis, such as motor neurons of the hindbrain and spinal cord.

These events are followed by midline prosencephalic development with formation of the commissural, chiasmatic, and hypothalamic plates, giving rise to the corpus callosum and septum pellucidum, optic nerve chiasm, and the hypothalamus, respectively. The earliest component of the corpus callosum appears at approximately 9 weeks, and by 12 weeks an independent corpus callosum is definable at the commissural plate. Axonal guidance following preexisting axon tracts from pioneering axons is critical in the formation of the corpus callosum. Mutations in VAX1 , GAP43 , heparan sulfate genes, and other genes are involved in a common phenotype where cortical neurons fail to reach the midline but continue to grow in swirled bundles of axons called Probst bundles.

Glial structures such as the glial wedge, midline zipper glia, indusium griseum glia, and subcallosal glial sling are present at the midline and are necessary for corpus callosum formation. Guidance by the glial wedge occurs through Slit/Robo signaling. Callosal axons are attracted through Netrin family chemoattractants and then repelled from recrossing by Slit-activated Robo molecules. Dorsal to the developing corpus callosum, the glia of the indusium griseum express Slit2. Nuclear factor I family transcription factors appear to regulate the development of midline glia and commissural development. Multiple additional signaling molecules, including ephrins, semaphorins, and neuropilins, are also involved in axonal guidance and are critical for proper brain development. As for neuronal precursors, emerging evidence indicates that radial glia undergo patterning by SHH and other organizational cues to generate restricted domains of precursors for astrocytes and myelinating oligodendrocytes.