Neuroembryology

Gastrulation

Gastrulation is the birthday of the nervous system. It is not only the time that bilateral symmetry and the three axes are established in the body of all vertebrates, but also the time when a neuroepithelium can first be identified and distinguished from primitive germinal tissues. The traditional concept of three germ layers dates from gastrulation as well, but the convenient conception of all mature tissues having been derived from one of the three layers is probably more arbitrary than biologic because the neural crest forms tissues assigned to all three germinal layers and the expression of many families of genes does not respect these germinal boundaries and mediates the development of structures corresponding to all three.

In simple chordates, such as amphioxi and amphibians, gastrulation is the invagination of a spherical blastula. In birds and mammals, the blastula is collapsed as a flattened, bilayered disk, and gastrulation appears not as an invagination but as a groove between two ridges on one surface of this disk, called the primitive streak on the epiblast. In each embryo the primitive streak establishes the basic body plan of all vertebrates: a midline axis, bilateral symmetry, rostral and caudal ends, and dorsal and ventral surfaces.

As the primitive streak extends forward, cells aggregate at one end, a collection designated the primitive node or Hensen node . Cells of the epiblast on either side move toward the primitive streak, stream through it, and emerge beneath it to pass into the narrow cavity between the two sheets of cells, with the epiblast above and the hypoblast below; these migratory cells give rise to the mesoderm and endoderm internally, and some then replace the hypoblast.

After extending about halfway across the blastoderm (epiblast), the primitive streak with the Hensen node reverses the direction of its growth and retreats, moving posteriorly as the head fold and neural plate form anterior to the Hensen node. As the node regresses, a notochordal process develops in the area rostral to it, and somites begin to form on either side of the notochord, with the more caudal somites differentiating first and successive ones differentiating anterior to the somites already formed. The notochord induces epiblast cells to form neuroectoderm (see “Induction”). Several genes essential in creating the fundamental architecture of the embryo and its nervous system are already expressed in the primitive node, and many reappear later to influence more advanced stages of ontogenesis.

Induction

Induction refers to the influence of one embryonic tissue on another such that both the inducer and the induced differentiate as different mature tissues. In the case of the nervous system, neural tube development may be defined in terms of gradients of inductive influences. Induction usually occurs between germ layers, as with the notochord (mesoderm) inducing the floor plate of the neural tube (ectoderm), although induction also may occur within a single germ layer. An example is the optic cup (neuroectoderm) inducing the formation of a lens and cornea from the overlying epithelium (surface ectoderm) that otherwise would have differentiated as more epidermis. Neural induction is the differentiation or maturation of neural structures from undifferentiated ectodermal cells as a result of the influence of surrounding embryonic tissues.

Induction was discovered in 1924, when Hans Spemann and Hilde Mangold demonstrated that the dorsal lip of the newt gastrula was capable of inducing the formation of an ectopic second nervous system when transplanted to another site in a host embryo, into another individual of the same species, or to a ventral site of the same embryo. This dorsal lip of the amphibian gastrula, also called the Spemann organizer, is homologous with the Hensen node of embryonic birds and mammals.

The first gene isolated from the Spemann organizer was Gsc (goosecoid), which encodes a homeodomain protein (see the later section “Transcription Factors and Homeoboxes”) able to recapitulate transplantation of the dorsal lip tissue when injected into an ectopic site. It also normally induces the prechordal mesoderm and contributes to prosencephalic differentiation. ^{, ,} In the Hensen node in the chick, even before the primitive streak is fully formed, Wnt8c is expressed and is essential for the regulation of axis formation and later for hindbrain patterning in the region of the future rhombomere 4 (r4). The regulatory gene Cnot, with major domains in the primitive node, notochord, and prenodal and postnodal neural plate, is also involved in the induction of prechordal mesoderm and in formation of the notochord in particular.

Cranial neural placodes are focal thickenings in the ectoderm in the head of vertebrate embryos that give rise to a wide variety of cellular types, including elements of the paired sense organs and neurons in cranial nerve sensory ganglia; the neural crest is the basis for the formation of these placodes. Ectodermal placodes (e.g., olfactory, ocular, otic) have gaps in the basal lamina through which presumptive neural crest cells delaminate and initiate peripheral migration. Neural crest tissue originates from the margins of the neural folds and from the dorsal midline with closure of the neural tube, from which it migrates into the periphery of the body in a predictable, programmed manner. Neural crest foci are in each of the primitive neural vesicles: prosencephalic neural crest originates at the dorsal part of the lamina terminalis and its cells migrate as a vertical sheet rostrally in the midline of the face; mesencephalic neural crest arises in the dorsal midline of the midbrain tegmentum and migrates mainly as three parallel streams of cells in the horizontal plane to induce most of the craniofacial structures of the orbits, membranous bone of the cranial vault, cartilages, most of the globe of the eye, blood vessels, nerve sheaths, melanocytes, adipose and collagenous connective tissue, and other structures. Rhombencephalic neural crest migrates from the dorsal midline of the hindbrain and spinal cord; a specialized part of the rhombencephalic neural crest is the cardiac neural crest that forms the Purkinje conduction system, septum, valves, and some other structures of the heart. Neural crest induction is a multigenetic process. ^,

The specificity of induction lies not in the inductive molecule but rather in the receptor in the induced cell. This distinction is important because foreign molecules similar in structure to the natural inductor molecule may sometimes be erroneously recognized by the receptor as identical; such foreign molecules may act as teratogens if the embryo is exposed to such a toxin. Induction occurs during a very precise temporal window; the period of responsiveness of the induced cell is designated its competence, and the cell is incapable of responding before or after that precise time.

Induction receptors are not necessarily in or on the plasma membrane of the cell but may be in the cytoplasm or in the nucleus. Retinoic acid is an example of a nuclear inducer. In some cases, the stimulus acts exclusively at the plasma membrane of target cells and does not require actual penetration of the cell. ^, The receptors that represent the specificity of induction are also genetically programmed. Notch is a particularly important gene in regulating the competence of a cell to respond to inductive cues from within the neural tube and from surrounding embryonic tissues. Some mesodermal tissues, such as smooth muscle of the fetal gut, can act as mitogens on the neuroepithelium by increasing the rate of cellular proliferation, ^, but this phenomenon is not true neural induction because the proliferating cells do not differentiate or mature. Some organizer and regulatory genes of the nervous system, such as Wnt1, also exhibit mitogenic effects, and insulin-like growth factor and basic fibroblast growth factor (FGF) act as mitogens, as well.

Early formation of the neural plate is not accomplished exclusively by mitotic proliferation of neuroepithelial cells; surrounding cells are also converted to a neural fate. In amphibians a gene known as Xash (achaete-scute) is expressed very early in the dorsal part of the embryo from the time of gastrulation and acts as a molecular switch to change the fate of undifferentiated cells to become neuroepithelium rather than surface ectodermal or mesodermal tissues. Some cells differentiate as specific types because they are actively inhibited from differentiating into others. All ectodermal cells are preprogrammed to form neuroepithelium, and neuroepithelial cells are preprogrammed to become neurons if not inhibited by genes that direct them along a different lineage, such as epidermal, glial, or ependymal.

The neural tube induces craniofacial development and mediates it through the neural crest, which migrates rostrally from the prosencephalon, at the dorsal part of the lamina terminalis, and from the dorsal midline of the mesencephalon. The prosencephalic neural crest migrates as a vertical sheet of cells in the midline of the future nose and forehead and forms, among other structures, the intercanthal ligament that hold the orbits together so that the eyes are directed forward in the face instead of being located at the sides of the head. This program is genetically determined in some families of mammals, including primates, felines, canines, bears, and koalas, as well as in one family of birds only, the owls. Other animals have laterally placed eyes, which provide better panoramic, but not stereoscopic vision.

Neurulation

Bending of the neural placode to form the neural tube requires extrinsic and intrinsic mechanical forces in addition to dorsalizing and ventralizing genetic influences, which are discussed in detail later in this chapter.

These forces arise in part from growth of the surrounding mesodermal tissues on either side of the neural tube, the future somites ( Box 63.1 ).

After surgical removal of mesoderm and endoderm from one side of the neuroepithelium in experimental animals, the neural tube still closes, but it is rotated and becomes asymmetrical. The mesoderm appears to be important for orientation but not for closure of the neural tube. Expansion of the surface epithelium of the embryo is the principal extrinsic force for folding of the neuroepithelium to form the neural tube. Cells of the neural placode are mobile and migrate beneath the surface ectoderm, which causes the lateral margins of the placode to become raised toward the dorsal midline. Growth of the whole embryo itself does not appear to be an important factor because neurulation proceeds equally well in anamniotes (e.g., amphibians), which do not grow during this period, and in amniotes (e.g., mammals), which grow rapidly at this time. FLOAT NOT FOUND

Among the intrinsic forces of the neuroepithelium, the cells of the floor plate have a wedge shape—narrow at the apex and broad at the base—that facilitates bending. Although the width of the floor plate is small, its site in the ventral midline is crucial and sufficient to allow a significant influence. It represents yet another aspect of induction of the floor plate by the notochord, apart from its influence on the differentiation of neural cells. The ependymal cells that form the floor plate are the first neural cells to differentiate, and they induce growth of the parenchyma of the ventral zone more than the dorsal regions. ^, This mechanical effect may also facilitate curving of the neural placode. The direction of proliferation of new cells in the mitotic cycle, determined in part by the orientation of the mitotic spindle, becomes another mechanical force shaping the neural tube. ^, Adhesion molecules are also probably an important mechanical factor for neurulation. In later stages, the ependymal cell–lined central canal, which is much larger in the fetus than in the newborn, may have a role in exerting a centrifugal force to create the tubular shape, although in early spinal cord development the central canal is a tall, narrow, midline slit and only later in fetal life does it assume a rounded contour as seen in transverse sections.

Neuroepithelial cells of the neural placode or plate downregulate the polarity of their plasma membrane so that the apical and basilar surfaces are not as distinct before neural tube closure. Cell differentiation in general involves such changes in cell polarity. The rostrocaudal orientation of most mitotic spindles of the neuroepithelium and the direction in which they push past the mass of daughter cells that they form also influence the shape of the neural tube ( Fig. 63.2 ).

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The neural tube closes in the dorsal midline first in the cervical region, with the closure then extending rostrally and caudally such that the anterior neuropore of the human embryo closes at 24 days and the posterior neuropore closes at 28 days, with the distances from the cervical region being unequal. This traditional view of a continuous zipper-like closure is an oversimplification. In the mouse embryo the neural tube closes in the cranial region at four distinct sites, with the closure proceeding bidirectionally or unidirectionally and in general synchrony with somite formation. ^, An intermittent pattern of anterior neural tube closure involving multiple sites has also been described in human embryos. In this closure pattern the principal rostral neuropore closes bidirectionally to form the lamina terminalis, an essential primordium of the forebrain.

Bending of the neural plate to form the neural tube is termed primary neurulation. Failure of the anterior neuropore to close by 24 days results in anencephaly. Because the lamina terminalis does not form, its derivatives (including the basal ganglia and other forebrain structures) do not develop. The lack of forebrain neuroectoderm results in failure of induction of the overlying mesoderm, and the cranium, meninges, and scalp fail to close in the midline. The term secondary neurulation refers only to the most caudal part of the spinal cord (i.e., conus medullaris), which develops from neuroepithelium caudal to the site of posterior neuropore closure. More details on abnormalities that occur because of problems with secondary neurulation are offered in other chapters in this textbook.

Neural crest cells arise from the dorsal midline of the neural tube at or shortly after the time of closure and migrate extensively along prescribed routes through the embryo to differentiate as the peripheral nervous system. This includes the dorsal root and sympathetic ganglia, adrenal medulla and carotid body chromaffin cells, melanocytes, and a few other cell types of ectodermal and mesodermal origin. ^,

Segmentation and Regionalization

Segmentation of the neural tube creates intrinsic compartments that restrict the movement of cells by physical and chemical boundaries between adjacent compartments. These embryonic compartments are known as neuromeres. Hindbrain neuromeres, known by the more specific term rhombomeres, are well established except for the inclusion of the spinal cord. Forebrain neuromeres of the diencephalon and telencephalon are known as prosomeres and traditionally have been considered simply as a continuation rostral to the notochord of the same type of segmentation in the longitudinal axis of the neural tube as was described by Herrick in 1910 and repeated in his monograph of 1948. More recent reconsideration of the developing forebrain has led to new proposals of its segmentation, however. The new considerations for prosomeres is the vertical axis of dorsoventral and ventrodorsal genetic gradients in the neural tube, in addition to the classical longitudinal axis divisions, as well as forebrain flexures.

The spinal cord superficially appears to be highly segmented; however, it is not intrinsically segmented in the embryo, fetus, or adult but rather corresponds in its entirety to the most caudal of the eight neuromeres that create the hindbrain. The apparent segmentation of the spinal cord results from clustering of nerve roots imposed by true segmentation of surrounding tissues derived from the mesoderm, tissues that form the neural arches of the vertebrae, somites, and associated structures. Regional differences already are known in the central autonomic neuronal clusters of the cervicosacral parasympathetic and thoracolumbar sympathetic centers. Differences in genetic expression are demonstrated between different adjacent parts of the spinal cord, which further confirms that the intrinsic spinal cord indeed is a segmented structure and not simply a long continuation of rhombomere 8. These segments are redesignated myelomeres .

Neuromeres of the hindbrain are designated rhombomeres. The entire cerebellar cortex, vermis, flocculonodular lobe, and lateral hemispheres develop from rhombomere 1 (r1), with a small contribution to the anterior vermis from the mesencephalic neuromere, but the dentate and other deep cerebellar nuclei are formed in rhombomere 2 (r2). ^, The rostral end of the neural tube forms a mesencephalic neuromere and six forebrain neuromeres by the Herrick scheme of the last century (i.e., two diencephalic and four telencephalic prosomeres) with further subdivisions. The segmentation of the human embryonic brain into neuromeres is summarized in Table 63.2 .

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The segments of the embryonic neural tube are distinguished by physical barriers formed by processes of early specializing cells that resemble the radial glial cells that appear later in development ^, and by chemical barriers from secreted molecules that repel migratory cells. Cell adhesion is increased in the boundary zones between rhombomeres, which also contributes to the creation of barriers against cellular migration in the longitudinal axis. Limited mitotic proliferation of the neuroepithelium occurs in the boundary zones between rhombomeres. Although cells still divide in this zone, their nuclei remain near the ventricle during the mitotic cycle and do not move as far centrifugally within the elongated cell cytoplasm during the interkinetic gap phases as they generally do. The rhombomeres of the brainstem may also be visualized as a series of transverse ridges and grooves on the dorsal surface, the future floor of the fourth ventricle; these ridges are gross morphologic markers of the hindbrain compartments. ^,

The first evidence of segmentation is a boundary that separates the future mesencephalic neuromere from r1 of the hindbrain. More genes play a role in this initial segmentation of the neural tube than in any boundaries that subsequently form to separate other neuromeres. The mesencephalic-metencephalic region appears to develop early as a single, independent unit or “organizer” for other neuromeres rostral and caudal to that zone. ^, The organizer genes recognized at the mesencephalic-metencephalic boundary for this earliest segmentation of the neural tube include Pax2, Wnt1, En1, En2, Pax5, Pax8, Otx1, Otx2, Gbx2, Nkx2-2, and fibroblast growth factor Fgf8. FGF signaling controls the somite boundary position for spatiotemporal HOX gene activation for segmentation of the somites, ventral neural tube patterning, and other structures. ^, Somite segmentation is initiated by Mesp2 via the Notch signaling pathway.

The earliest known gene with regional expression in the mouse is Pax2, and it is expressed even before the neural plate forms. It is the earliest gene recognized in the presumptive region of the midbrain-hindbrain boundary. ^, In invertebrates, Pax2 is important for the activation of Wg (wingless) genes; this relationship is relevant because the first gene definitely associated with an identified midbrain-hindbrain boundary in vertebrates is Wnt1, a homologue of Wg. Regulation of Wnt1 may be divided into two phases. In the early phase (one or two somites), the mesencephalon broadly expresses the gene throughout; in the later phase (15–20 somites), expression is restricted to the dorsal regions, the roof plate of the caudal diencephalon, the mesencephalon, the myelencephalon, and the spinal cord, but it is also expressed in a ring that extends ventrally just rostral to the midbrain-hindbrain boundary and in the ventral midline of the caudal diencephalon and mesencephalon. Wnt1 is essential in activating and preserving the function of the mouse engrailed genes En1 and En2. En1 is coexpressed with Wnt1 at the one-somite stage in a domain only slightly caudal to Wnt1, which includes the midbrain and r1, the rostral half of the pons, and the cerebellar cortex but excludes the diencephalon. Activation of En2 begins at the four-somite stage, and its function in mesencephalic and r1 development is similar, with differences in some details, particularly their roles in cerebellar development. ^, The homeobox gene Otx2 appears early in the initial boundary zone of the midbrain-hindbrain, and as with Wnt1, it appears to be essential for the later expression of En1, En2, and Wnt1. ^,

The creation of neuromeres allows the development of structures within regions of the brain without the wandering of neuroblasts that form these nuclei to other parts of the neuraxis where they would not be able to later establish their required synaptic relationships. The interaction of genes with one another is a complexity that makes analysis of single-gene expression more difficult in interpreting programmed malformations of the brain.

Patterning of the Neural Tube

Development of the basic characteristics of the body plan is called patterning. These patterns are the anatomic expression of the genetic code within the nuclear DNA of every cell, but they may also result from signals from neighboring cells carried by molecules that are secretory translation products of various families of organizer genes, each in a highly precise and predictable temporal and spatial distribution.

Early development of the CNS in all vertebrates, even before closure of the neural placode or plate to form the neural tube, requires the establishment of a fundamental body plan of bilateral symmetry, with cephalization, or the identity of head and tail ends, and determination of the dorsal and ventral surfaces. These axes of the body itself and the CNS require the expression of genes that impose gradients of differentiation and growth. The genes that determine the polarity and gradients of the anatomic axes are called organizer genes. Many express themselves in the CNS and in other organs and tissues. ^, The bilateral symmetry of many organs and programmed asymmetries, probably including neural structures such as the different targets of the left and right vagal nerves and left-right asymmetries in the cerebral cortex, are determined in large part by Pitx2, a gene expressed as early as in the primitive node. Some genes function to stimulate or inhibit the expression of others, or there is an antagonism or equilibrium between certain families of genes, as exemplified by those that exert dorsoventral or ventrodorsal gradients. The difference between an organizer gene and a regulator gene is its function, and the same gene often subserves both roles at different stages of development. The definitions and programs of these two groups are summarized in Box 63.2 .

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