Building Brains: Chapter 2 - Developing Nervous Systems PDF

The Anatomy of Developing Nervous Systems 2 An understanding of the mechanisms of neural development is built on a knowledge of the anatomy of developing nervous systems. There is a vast amount of literature descri...

The Anatomy of Developing Nervous Systems 2 An understanding of the mechanisms of neural development is built on a knowledge of the anatomy of developing nervous systems. There is a vast amount of literature describing the three-dimensional geome- try of developing neural structures from a plethora of species and here we shall give only the briefest of accounts containing essential details required to follow later chapters. The focus is on the model organisms described in Chapter 1. Nervous systems develop both peripheral and central components. The periphery extracts information from around and within the body. It passes neural signals to the centre, where neural information is proc- essed and memories are stored. Decisions made centrally are signalled back to the periphery to make the body respond. The most complex nervous systems are found in vertebrates, in which the brain and spinal cord make up the central nervous system, or CNS, and all the neurons that reside or extend axons outside the CNS make up the Neuroectoderm peripheral nervous system, or PNS. Ectoderm Mesoderm 2.1 The nervous system develops from the embryonic neuroectoderm Endoderm One feature common to the embryos of animals is their early organiza- tion into three primary tissues called the germ layers (from the Latin word germen, meaning seed or bud). These layers are shown on the drawing of a frog embryo cut in half on the right: (i) an outer layer called the ectoderm (yellow and orange); (ii) a middle layer called the mesoderm (purple); (iii) an inner layer called the endoderm (grey). Ectodermal derivatives include the epidermis (or skin) and the nervous system, which is derived from the neuroectoderm (i.e. the neurogenic region of the ectoderm, shown in orange). The neuroectoderm is some- epithelium a tissue that lines the times called the neuroepithelium since it comprises cells on the external and internal surfaces, includ- surface of the embryo that are part of the epithelium. The colour ing internal cavities and organs and scheme on the right is used throughout this chapter (and where it other free open surfaces of the body, would be useful in later ones) to help the reader recognize the germ of all animals and their immature developing forms. Building Brains: An Introduction to Neural Development, First Edition. David Price, Andrew Jarman, John Mason and Peter Kind. # 2011 John Wiley & Sons, Ltd. Published 2011 by John Wiley & Sons, Ltd. 20  THE ANATOMY OF DEVELOPING NERVOUS SYSTEMS layers and their derivatives. Mesodermal derivatives include muscles and skeleton and endodermal derivatives include gut and associated organs. There is great variation from species to species in the shapes of the three germ layers but their presence and relative positions remain conserved. Formation of the germ layers follows the repeated division of a fertil- ized egg to generate a collection of cells that are then rearranged to generate the germ layers. This rearrangement is called gastrulation. Gastrulation involves the movement of cells from the outer surface of the embryo to its inside. While this feature of gastrulation is common to the embryos of different species, the exact three-dimensional anat- omy of gastrulating embryos varies greatly between different species, as will be seen from the examples in this chapter. The sequence shown on the left illustrates gastrulation in cross sections through a Drosophila embryo. We do not know for sure what mechanisms make cells move into the embryo but it is likely that changes in the shapes of cells gen- erate mechanical forces that are important. In the diagram on the left, some of the cells at the bottom of the embryo become narrower along their outer edge, as if pulled by a drawstring, effectively pinching this surface. The cells caught in the pinch move inwards. Such changes in shape are brought about by contractile proteins in cells (the types of protein that do this are described throughout Chapters 6 to 8). Other forces from outside these cells pulling them into the embryo might also be important. 2.2 Anatomical terms used to describe locations in embryos The aim of this chapter is to describe the major stages of develop- ment of the nervous systems of the model organisms described in Chapter 1. Before doing so, we need to explain some terms used to Dorsal describe how the different parts of developing organisms are lo- cated relative to each other; these same terms are also applied to adult organisms. The terms front, back, top, bottom and sides are avoided since they are likely to be used inconsistently in different Lateral species (e.g. along the top of a fish we might expect to find its spinal Anterior Posterior cord whereas at the top of a person we might expect to find a head). or Medial or Rostral Caudal The end of the embryo where the head forms is referred to as Lateral anterior, and its opposite end is posterior. Anterior is also described as rostral (which means ‘pertaining to a beak’); posterior is also described as caudal, (which means ‘pertaining to a tail’). The axis running through the embryo from anterior to posterior is referred to as the anteroposterior or rostrocaudal axis. Perpendicu- lar to this axis is the dorsoventral axis, running from the embryo’s Ventral dorsal side (i.e. its back, where the spinal cord develops in verte- brates) to its ventral side (meaning the side towards the chest). The third axis (mediolateral) runs perpendicular to the other two from medial (towards the midline) to lateral (away from the midline). These axes are indicated for an adult organism and an embryo in the drawings on the left.  DEVELOPMENT OF THE NEUROECTODERM OF INVERTEBRATES  21 2.3 Development of the neuroectoderm of invertebrates 2.3.1 C. elegans The embryo’s first cleavage generates a large cell (called the AB blasto- blastomere any of the cells resulting mere) and a smaller cell (called the P1 cell). The AB blastomere gives from the first few cleavages of a fertil- rise to ectodermal cells that spread to form the outer wall of the ized egg during early embryonic embryo, originally called the hypodermis but now sometimes called development. the epidermis, and the nervous system. The nervous system arises al- most entirely from the AB blastomere. Gastrulation in C. elegans is not epidermis the outermost layers of cells covering the exterior body as spectacular as in other species in which cells move over much surface. greater distances, but has the equally important result of internalizing cells that form endoderm and mesoderm. These cells come from the P1 cell and generate internal structures of the embryo, including the muscles, gut and gonads. These events are summarized in Fig. 2.1(a). To generate the nervous system, some of the ectodermal cells situ- ated ventrally migrate inside the embryo (red arrows Fig. 2.1(b)) where they differentiate to form neurons (Fig. 2.1(c)). A fully developed C. ele- gans has 302 neurons with about 7000 synapses and 56 glial cells. Many of its neurons are organized into dorsal and ventral nerve cords running along the length of the worm. C. elegans has no real brain, but dense col- lections, or ganglia, of sensory neurons and interneurons are present in the anterior of the organism. There are also smaller ganglia in the tail. The nerve cords link these anterior and posterior ganglia; they contain motor neurons and receive sensory inputs. An excellent website (Wor- matlas) contains more detailed information.1 2.3.2 Drosophila The early stages of development involve an unusual process of nuclear replication without cell division. This results in the formation of a nu- clear syncytium, the name given to a structure in which many nuclei share a common cytoplasm. This syncytial stage is important because it provides nuclei the opportunity to communicate without cell membranes getting in the way (discussed further in Chapter 4). As the time of gastrulation approaches, several hours after fertilization, dividing nuclei move out to the surface of the embryo and become sur- rounded by membranes, forming cells. The embryo is now a cellular blastoderm. Fig. 2.2 picks up from this point in development. blastoderm the superficial layer of Gastrulation gets underway with the formation of a furrow along the early embryo in species whose the embryo’s ventral surface. Future mesodermal cells enter the inte- eggs contain relatively large amounts rior (Fig. 2.2(a)). As this is happening, ectodermal cells that were ini- of yolk; cell division occurs in this tially positioned laterally along the two sides of the ventral part of layer, which surrounds the yolk in insects but is a flat disc at one pole of the embryo now come together at the ventral midline (indicated by the egg in birds. the arrows in Fig. 2.2(a)). These ventral ectodermal cells become the neuroectoderm, which goes on to form the fly’s CNS. The formation of the CNS involves cells in the neuroectoderm enlarging and mov- ing inside the embryo, a process called delamination, where they 1 http://www.wormatlas.org/ [20 November 2010]. 22  THE ANATOMY OF DEVELOPING NERVOUS SYSTEMS Zygote (P0) (a) AB P1 EMS P2 AB AB AB AB C MS E P3 C MS E P3 Ectoderm: hypodermis nervous system Muscles, gut, gonad, germline (b) Endoderm Hypodermis Mesoderm (c) Sensory neuron motor neuron Fig. 2.1 Key stages in the development of C. elegans: development is highly stereotypical and the lineages of all the cells of C. elegans are known. (a) This diagram charts the first divisions of the embryo. The nervous system arises from the AB blastomere. Gastrulation is complete once derivatives of the AB blastomere have covered the internalized derivatives of the P1 cell. (b) Arrows show the inward migration of AB-derived ectodermal cells on the ventral side of the embryo. (c) AB-derived ectodermal cells form sensory and motor neurons along the body: single examples of each are shown here.  DEVELOPMENT OF THE NEUROECTODERM OF INVERTEBRATES  23 (a) GASTRULATION Anterior Dorsal Ectoderm Mesoderm Neuroectoderm Posterior Mesoderm Ventral Neuroectoderm (b) Epidermis DELAMINATION Mesoderm Sense organ precursor Neuroblasts Neuroblasts (c) CNS FORMATION Ganglion Neurons/glia mother Neuroblast cell (d) time AXONAL DEVELOPMENT Longitudinal Neuron fascicles Afferent nerves Efferent nerves Ventral nerve cord Commissures Fig. 2.2 Major stages of Drosophila development. (a) Blastoderm stage: arrows show the main directions of cell movements during gastrulation. The neuroectoderm (orange) is initially split into two domains along either side of the ventral part of the embryo and as the mesoderm involutes these domains coalesce ventrally. (b) Subsequently, some of the cells in this ventral neurogenic region move inside the embryo to become neuroblasts; this process is called delamination. Other cells in the lateral ectoderm delaminate to become sense organ precursors: the development of these cells is shown down the left between (b) and (d) (see also Section 2.6.1). (c) The neuroblasts divide to generate the neurons and glia of the Drosophila’s CNS, many of which reside in the ventral nerve cord. This is achieved through the production of intermediate cells the ganglion mother cells (GMCs), which divide to generate pairs of neurons or glia. (d) Sensory nerves from developing sense organs converge on the ventral nerve cord, which generates motor nerves. Axons in the ventral nerve cord are organized into longitudinal fascicles linked by commissures. 24  THE ANATOMY OF DEVELOPING NERVOUS SYSTEMS form neuroblasts (Fig. 2.2(b)). Neuroblasts are dividing cells that generate neurons and glia. The ectoderm becomes divided into reiterated units called seg- ments. Within each segment, five waves of delamination eventually result in a stereotypic array of some 60 neuroblasts. Neuroblasts are progenitor cells that divide many times to form numerous intermedi- ate cells called ganglion mother cells (GMCs) (Fig. 2.2(c)). Each GMC di- vides just once more to form neurons and glia. GMCs and their progeny pile up on the neuroblasts forming the bilaterally symmetrical ventral nerve cord. Eventually, each segmental unit of the larval CNS (a ganglion) con- tains about 800 neurons, giving a late embryonic CNS consisting fascicle a bundle of nerve or muscle of about 100 000 neurons in total. A scaffold of axon bundles is gener- fibres. ated on the inner (or dorsal) surface of the ventral nerve cord. Some bundles run in the anteroposterior direction and are called longitudi- commissure a bundle of axons (commissural axons) that extends nal fascicles while others run perpendicular to them and are called across the midline to connect struc- transverse commissures (Fig. 2.2(d)). The development of peripheral tures on either side of the nervous sensory organs depicted in Fig. 2.2 will be discussed again below, in system. Commissures are important Section 2.6. A second stage of neurogenesis occurs during metamor- for coordinating neural activity on the phosis in the pupa. This gives rise to a more complex adult nervous two sides of the animal. system with many more neurons.2 2.4 Development of the neuroectoderm of vertebrates and the process of neurulation Ventricles In mature vertebrates, a narrow cavity filled with cerebrospinal fluid called the central canal runs longitudinally through the middle of the spinal cord. It expands into several larger cavities called ventricles inside the brain (left). The fact that the mature central nervous system is hol- low reflects its origin from a hollow structure in the embryo known as the neural tube. The process by which the neural tube is formed and acquires increasingly complex morphology is referred to as neurula- tion; it occurs through (i) rapid growth at rates that vary from region to region, (ii) cell movements and (iii) changes in cell shapes. Neurulation is usually considered to be of two types, primary and secondary. Primary neurulation is the process by which a sheet of neuroectoderm called the neural plate rolls up as it grows to form Central the neural tube. This is schematized in Fig. 2.3, where the neuroecto- canal derm (orange region situated dorsally) of a simplified vertebrate embryo (in this case a frog) is shown in isolation. The buckling that occurs as the neural plate rolls up is associated with changes in the shape of its cells: in general, they become more columnar but in some regions, such as along the centre of the neural plate, there are more complex shape changes. It is hard to tell whether changes in the shapes of cells are the cause or the effect of changes in the shape of the neural plate; the mechanics of neural tube closure remain poorly understood. Secondary neurulation achieves the same result as primary neurulation, that is a tube of neural tissue, but through a 2 For details refer to Tissot, M. and Stocker, R. F. (2000) Metamorphosis in Drosophila and other insects: the fate of neurons throughout the stages. Prog Neurobiol., 62,89–111.  DEVELOPMENT OF THE NEUROECTODERM OF VERTEBRATES AND THE PROCESS OF NEURULATION  25 Dorsal (D) Anterior (A) Posterior (P) Ventral (V) A Fig. 2.3 Schematic of primary neuru- lation in a vertebrate: note how the lat- A D eral edges of the neural plate roll up and join dorsally, while cells that are medial in the neural plate end up ven- V trally. This establishes the dorso- Lateral ventral axis of the vertebrate neural Medial P P tube. different process that involves the hollowing out of an initially solid rod of tissue (see below, Section 2.5). 2.4.1 Frog The first steps in development are illustrated in Fig. 2.4(a). As cell divi- sion progresses, the embryo’s dorsal region, called the animal cap, becomes packed with many small cells. Cells in the opposite ventral region are fewer, larger and yolk-rich (this region is called the vegetal hemisphere). Prior to gastrulation, a fluid-filled cavity known as the blastocoel opens beneath the animal cap; the embryo is now called a blastula. Fig. 2.4(b) illustrates gastrulation, which involves the inward movement of cells from the embryo’s outer layer at a region called the blastopore. This involution of a layer of cells is like the deformation of the surface of a balloon when poked. The first cells to move in migrate anteriorly from the dorsal lip of the blastopore (which has important organizing functions, discussed in Chapters 3 and 4). Their movement displaces the blastocoel anteriorly (it eventually disap- pears). These cells form the endoderm which lines the primitive gut (called the archenteron), the mesoderm of the future head and a tran- sient dorsal mesodermal structure, the notochord, which is essential for differentiation of the overlying neuroectoderm. During these events, cells derived from the animal pole spread to cover the embryo with ectoderm (a process called epiboly). The result is an embryo surrounded by ectoderm, with an inner primitive gut lined with endoderm and with mesoderm forming between them. Fig. 2.4(c) illustrates subsequent major steps in neural development, including primary neurulation. The development of the neural crest along the dorsal aspect of the neural tube is discussed further 26  THE ANATOMY OF DEVELOPING NERVOUS SYSTEMS Blastocoel (a) Animal cap Archenteron Blastula (b) Neuroectoderm Dorsal Mesoderm GASTRULATION Anterior Endoderm Posterior Blastopore Ventral Blastopore (c) Neural plate Endoderm Ectoderm Anterior Posterior Mesoderm Ectoderm Neural plate Mesoderm Archenteron Neural tube Optic tectum Notochord Somites Neural crest Lens Neural tube Optic Retinal chiasm Retina ganglion cell Fig. 2.4 The main stages of development of Xenopus: (a) formation of the blastula, (b) gastrulation and (c) development of the neural plate and neural tube (orange) through primary neurulation. In (c), the neural plate is first shown in a transected embryo tilted towards the viewer along its anteroposterior axis. Cells along the lateral edges of the neural plate (brown) form the neural crest: its cells migrate laterally throughout the body (see later in this chapter, Section 2.6). The final drawings show the tadpole stage: brain structures such as the retinae and optic tectum, which are major components of the visual system, develop from the neural tube anteriorly and become connected by axons, forming a favourite model for the study of axonal guidance (discussed further in Chapter 8).  DEVELOPMENT OF THE NEUROECTODERM OF VERTEBRATES AND THE PROCESS OF NEURULATION  27 below in the context of the formation of the peripheral nervous system (Section 2.6). 2.4.2 Chick When the egg is laid the embryo is a disc of cells on the surface of the yolk. It comprises a central transparent area (the area pellucida) surrounded by an opaque ring (the area opaca) (Fig. 2.5(a)). The area pellucida is transparent because there is a fluid-filled cavity, initially cell-free, between the yolk and an overlying single-cell-thick layer of epithelial cells. The epithelial layer is called the epiblast. The area epiblast the layer of cells in the early opaca is opaque because cells lie beneath the epiblast in contact with embryos of birds, reptiles and mam- the yolk and there is no cavity. The epiblast generates the three germ mals that gives rise to the three germ layers of the embryo. layers at gastrulation. The posterior part of the area pellucida contains a crescent- shaped ridge of small cells called Koller’s sickle (Fig. 2.5(a)). Fig. 2.5 (b) shows the remarkable events that occur in this region. As epi- blast cells are produced they move posteriorly in the plane of the epiblast towards Koller’s sickle, from where they move anteriorly along the midline forming a structure called the primitive streak. Formation of the primitive streak involves the ingression of epiblast cells into the interior of the embryo and results in the generation of the germ layers of the embryo, that is gastrulation (Fig. 2.5(c)). As the primitive streak lengthens from posterior to anterior, a bulge called Hensen’s node appears at the anterior tip of the streak. Hensen’s node is equivalent to the dorsal lip of the blastopore in Xenopus and has important organizing functions that will be dis- cussed in Chapter 3. Hensen’s node contains cells that are precursors of the notochord. The notochord is a midline mesodermal structure that has been described above in the context of Xenopus development (Fig. 2.4(c)). It is long and thin and it forms by a process of cell movement called conver- gent-extension in which tissue elongation is achieved by the conver- gence and intercalation of adjacent cells in a sheet to form a narrower, longer strip of tissue, a principle illustrated on the right. This process forms the entire length of the notochord anterior to the primitive streak. Hensen’s node is sometimes described as retreating from the anterior end of the embryo, leaving behind the head process (cells that will eventually form the chick’s head) and subsequently cells destined to become the notochord. This relative movement of Hensen’s node is indicated by an arrow in the right-hand diagram in Fig. 2.5(c). With the retreat of Hensen’s node, structures begin to differentiate; since Hensen’s node retreats from the anterior end of the embryo first, development in this region is ahead of that in more posterior regions. The process of neurulation is similar to that of other vertebrates and is not repeated in Fig. 2.5. Fig. 2.5(d) shows later stages of development including (i) the elonga- tion and bending of the neural tube, (ii) the formation of anterior swellings that become the forebrain, midbrain and hindbrain (further information on these regions of the brain will be found in the following section on mouse development), (iii) the division of the 28  THE ANATOMY OF DEVELOPING NERVOUS SYSTEMS (a) Anterior Hypoblast islands Area pellucida Area Epiblast pellucida Koller’s sickle Koller’s sickle Egg Area opaca Area Posterior opaca (b) Hypoblast Primitive streak (c) Head Primitive Hensen’s node process streak Primitive streak Hensen’s node Primitive streak Epiblast (d) Midbrain Mesoderm Endoderm Rhombomeres r1 Forebrain Forebrain r2 r4 r6 Branchial Cervical r8 spinal arches cord Somites Limb bud Thoracic spinal cord Hensen’s node Somites Fig. 2.5 Main stages of chick development. (a) A small portion of the shell of the hen’s egg is removed to expose the early embryo (yellow), which is enlarged and viewed from above in the central drawing. A perpendicular section through the embryo’s posterior part reveals the cells beneath the epiblast around Koller’s sickle. (b) Arrows show the movement of cells in the epiblast posteriorly and then inside the embryo to form the primitive streak and, at its anterior end, Hensen’s node. (c) Gastrulation involves the movement of epiblast cells inside the embryo and the formation of the three germ layers, shown in a slice through the embryo. This is similar in principle to gastrulation in Xenopus but takes place in a flatter embryo. Hensen’s node retreats from the anterior end where the head process forms, elongating the neural plate as it moves. (d) Later stages show elongation of the nervous system, the formation of somites, the development of brain structures and the growth of nerves from the CNS (arrows). The branchial arches are a set of mesodermal structures on either side of the developing pharynx.  DEVELOPMENT OF THE NEUROECTODERM OF VERTEBRATES AND THE PROCESS OF NEURULATION  29 hindbrain into a series of anatomically distinguishable tissue blocks somites segmental masses of meso- called rhombomeres (see Chapter 4, Section 4.3.4) and (iv) the forma- derm lying on either side of the noto- tion of somites. chord and neural tube during the development of vertebrate embryos. 2.4.3 Mouse Following fertilization, the embryo develops through the blastocyst blastocyst the mammalian embryo stage (Fig. 2.6(a)) before implanting into the uterine wall and under- prior to gastrulation, comprising up to going gastrulation (Fig. 2.6(b)). At implantation, the mouse blastocyst about 100 cells surrounding a fluid- comprises three tissues: the epiblast, situated at one pole of the filled cavity. embryo, the primitive endoderm beneath it and the trophectoderm surrounding both tissues and the blastocoel cavity (which will disap- pear later). After implantation the epiblast expands and a central fluid-filled cav- ity opens in the middle of it. The epiblast organizes into an epithelium that surrounds this new cavity and is itself surrounded by primitive endoderm. The trophectoderm generates tissues such as the ectopla- cental cone and extraembryonic ectoderm that go on to form placenta and membranes surrounding the embryo. Whereas the chick epiblast is essentially a flat disc on the surface of the yolk, the mouse epiblast is a concave sheet on the inside of the embryo, but otherwise the process of gastrulation is similar in the two species. As in the developing chick embryo, gastrulation in the mouse embryo involves the movement of cells in the plane of the epiblast towards and through the primitive streak to form a new mesodermal layer between the outer endoderm and the inner ectoderm (shown in the right-hand drawing in Fig. 2.6(b)). The primitive streak elongates along the middle of the embryo in its anteroposterior axis (see Section 2.2 above for a reminder of the body axes). The node (which is equivalent to Hensen’s node in chick) forms at the anterior end of the primitive streak (Fig. 2.6(c)). The notochord grows anteriorly from the node forming a narrow midline rod ending in the broader prechordal mesoderm under the future forebrain. The notochord comes to lie along the midline of the embryo beneath the part of the ectoderm that will form the nervous system, the neuroectoderm (Fig. 2.6(c)). The process of primary neurulation begins in the overlying neuroec- toderm with the formation and folding of the neural plate (Fig. 2.6(c)) to form the neural tube. The neural plate is broader anteriorly and the folds it generates (the cranial neural folds, Fig. 2.6(d)) are larger than those made posteriorly. When they fuse, the cranial neural folds pro- duce anterior swellings of the neural tube (called vesicles) that will develop into the brain. Cells along the dorsal neural tube give rise to the neural crest (Fig. 2.6(d) and see Section 2.6), which forms at the junction of the surface ectoderm and the neuroectoderm. Strips of mesoderm on either side of the developing neural tube generate the somites (Fig. 2.6(d)), whose derivatives include vertebrae, ribs and skeletal muscle. Neural tube closure begins around the posterior boundary of the midbrain and spreads anteriorly and posteriorly from this point, as if a zip were being closed in both directions (Fig. 2.6(d)). While this is 30  THE ANATOMY OF DEVELOPING NERVOUS SYSTEMS (a) Blastocyst Epiblast Trophectoderm Primitive endoderm Blastocoel Ectoplacental (b) cone GASTRULATION Mesoderm Extraembryonic ectoderm Primitive streak Epiblast Endoderm Neural plate: Neuroectoderm Future forebrain (c) viewed from above Future midbrain Future hindbrain Neuroectoderm Primitive streak Notochord Future spinal cord Head Node process Notochord Neuroectoderm (d) turned end-on Floor Cranial plate neural folds Neural crest Somite Prechordal Primitive mesoderm streak Foregut Notochord Neuroectoderm Node Somite Roof plate Proliferation Neural crest Migration Optic cells sulcus Differentiation Somite Floor plate Notochord Fig. 2.6 Development of the mouse nervous system from (a) the blastocyst stage to (d) neural tube closure. (b) Cell movements at gastrulation, indicated by arrows, result in the formation of the primitive streak. To help understand this stage, imagine the flat chick embryo on the left in Figure 2.5(c) rolled up with its right- and left-hand edges joined and the primitive streak on the inside: essentially, this would give the diagram on the right in (b) here. (c) The notochord and the neuroectoderm form anterior to the node. Look at the diagram on the right in Figure 2.5(c): imagine looking at it along the surface of the page from its left-hand side, in which case the neuroectoderm (orange) would be to the left, the primitive streak to the right and the node in the middle, and if it is bent upwards at its ends it will have the equivalent layout of structures to those on the left of (c) and (d) here. The neural plate is divided into domains that will form the forebrain, midbrain and hindbrain before it folds as indicated by arrows. (d) The origin of the neural crest is shown. Growth of the neural tube is accomplished by proliferation on the side nearest the lumen, followed by migration to and differentiation on the other side. Further development continues in Figure 2.8.  DEVELOPMENT OF THE NEUROECTODERM OF VERTEBRATES AND THE PROCESS OF NEURULATION  31 underway, new closures occur further anteriorly and the closures spreading from these points meet to complete the formation of the neural tube. Photographs of neural tube closure are shown in Fig. 2.7. Analysis of the mechanisms regulating neural tube closure is very im- portant for humans. Our neural tube closes in very similar ways and defects of these processes are relatively common, for example generat- ing spina bifida (Box 2.1). At the stage of neural tube closure, the embryo alters its shape dra- matically. At the start of this phase, most of the anteroposterior axis of the closing neural tube is bent in a large curve with the convexity towards its ventral side: as shown in Fig. 2.6(d), it looks a bit like a snake rearing its head. Cell movements indicated by arrows in Fig. 2.8(a) cause it to turn. This process of turning results in its antero- posterior axis becoming concave along its ventral surface (Fig. 2.8(a)): effectively, the neural tube curls up into a shape retained until birth. The anterior part of the neural tube forms the forebrain (or prosen- cephalon), the midbrain (or mesencephalon) and the hindbrain (or rhombencephalon) (Fig. 2.8(a)). The optic vesicles form bilaterally and generate the retinae and optic nerves (development of the eye is outlined in more detail in Box 2.2). The posterior part of the neural tube forms the spinal cord. Fig. 2.8(b) shows more details of the embryonic brain. The fore- brain vesicle expands to form two bilateral telencephalic vesicles and a central diencephalic vesicle that becomes engulfed by the rapidly expanding telencephalic vesicles. The telencephalon is di- vided into dorsal and ventral components that differ anatomically and molecularly. Dorsal telencephalon generates the cerebral cor- tex, which includes the neocortex (the part of the cerebral cortex that has expanded massively in the evolution of higher mammals) and surrounding phylogenetically older cortical regions such as the hippocampus. Ventral telencephalon generates the basal ganglia (large groups of neurons lying under the cortex involved in the control of movement). The major derivative of the diencephalon is the thalamus, which transmits sensory input to the cerebral cortex. Throughout the process of primary neurulation, most prolifera- tion to generate new progenitors and/or neurons occurs on the inner side of the neural tube closest to its lumen (Figs 2.6(d) and 2.8(b)). Cells destined to become neurons migrate away from this prolifera- tive zone towards the outside of the tube to differentiate into ma- ture neurons. In the brain, the proliferative zone is known as the ventricular zone, since the lumen of the embryonic brain forms the ventricular system of the adult. In the cerebral cortex, the first cells to migrate and differentiate form a structure called the pre- plate, that is later split into a superficial layer called the marginal zone and a deep layer called the subplate by newly arriving neurons which form a rapidly thickening layer called the cortical plate (Fig. 2.8(b)). The marginal zone forms layer 1 of the cortex, the corti- cal plate forms layers 2 to 6, and the subplate is a transient structure that largely disappears after birth. These processes and structures will be discussed more in later chapters. 32  THE ANATOMY OF DEVELOPING NERVOUS SYSTEMS Fig. 2.7 Scanning electron micrographs of neural tube closure in mouse embryos: reprinted from Copp, A. J., Brook, F. A., Estibeiro, J. P., Shum, A. S. W. and Cockroft, D. L. (1990) The embryonic development of mammalian neural tube defects. Prog. Neurobiol., 35, 363–403 with permission from Elsevier. Abbreviations: hnf, mnf or fnf: hindbrain, midbrain or forebrain neural folds; cnt: caudal neural tube; pn, posterior neuropore; so, somite. Arrows indicate the directions of neural tube closure. Scale bars are 0.1–0.3 millimeter.  DEVELOPMENT OF THE NEUROECTODERM OF VERTEBRATES AND THE PROCESS OF NEURULATION  33 Box 2.1 Neural tube defects Neural tube defects are congenital malformations of the CNS resulting from a failure of neurulation. There are many different types, the commonest of which are (i) anencephaly, in which the cranial neural folds do not fuse in the developing embryo and most or all of the brain is missing, and (ii) spina bifida, in which there is a failure of the neural tube to fuse at its caudal end resulting in either an open lesion on the spine, with significant damage to the nerves and spinal cord, or a closed lesion. These two defects have a prevalence of about one in 1000 births, with variations throughout the world; anencephaly results in death around birth whereas infants with spina bifida can survive with a variable degree of disability. Spina bifida cystica is a severe condition causing nerve damage and disability, in which the membranes of the spinal cord and, rarely, spinal nerves, protrude through openings in the spine resulting in a sac filled with cerebrospinal fluid on the back. Spina bifida occulta is a mild condition in which the spinal cord is normal and there are no openings to the back, although there may be a gap in the vertebral column and subtle motor and sensory problems can develop with age. Craniorachischisis is a relatively rare failure of closure involving the entire body axis. The causes of neural tube defects are genetic and environmental, but are poorly understood.3 Anencephaly Craniorachischisis Neural folds Somites Spina bifida Caudal neuropore Neuropore an opening at one or 3 other end of the neural tube that nor- For more information see Copp, A. J. and Greene, N. D. (2010) Genetics and development of neural tube defects. J. Pathol., 220, 217–30. mally closes eventually 34  THE ANATOMY OF DEVELOPING NERVOUS SYSTEMS (a) Midbrain Forebrain Hindbrain Optic vesicle Hippocampus Neocortex Forebrain: (b) Basal Dorsal ganglia telencephalon Ventral Eye telencephalon Lens Ventral diencephalon Retina Optic Diencephalon stalk Telencephalon Marginal zone Midbrain Cortical plate Preplate Subplate Floor plate Hindbrain Ventricular zone (c) SI Visual cortex VIS Somatosensory cortex AUD SII Auditory cortex Fig. 2.8 Development of the mouse brain, continued from Figure 2.6(a) A reversal in the embryo’s shape is caused by move- ments indicated by thick arrows. The drawing at the top is a simplified version of the drawing in Figure 2.6(d). Drawing on the right shows more detail of formation of the forebrain, midbrain, hindbrain and optic vesicles. (b) Subsequent growth and special- ization increase brain complexity. Growth is achieved through continuing cell divisions in the brain’s ventricular zone followed by migration of these cells towards the outer surface of the brain where they differentiate (see also Fig. 5.16 for more detail). (c) The mature brain, containing maps of sensory surfaces such as the skin (more on this in Chapter 9).  DEVELOPMENT OF THE NEUROECTODERM OF VERTEBRATES AND THE PROCESS OF NEURULATION  35 Box 2.2 Development of the mammalian eye (a) As the cranial neural folds fuse (diagram taken from Fig. 2.6(a)), two pits appear bilaterally in the region destined to become forebrain. These pits are called the optic sulci (singular: sulcus). (b) The optic sulci con- tinue to deepen laterally and bulge from the two sides of the forebrain to become the optic vesicles. (c) The optic vesicles remain connected to the developing forebrain by the optic stalks, which will become the optic nerves, and approach the surface ectoderm, with which they interact. (d) The neuroectoderm under the sur- face ectoderm changes its shape forming the optic cup. The layer on the inside of the cup becomes the retina. (e) The lens forms from a vesicle derived from the surface ectoderm (called the lens placode, see Fig. 2.11) overlying the optic cup. (f) The mature eye. Optic sulcus (a) (b) Optic Neural folds sulcus Surface ectoderm Optic sulcus Neural folds Surface ectoderm (c) Optic Optic vesicle stalk Lens placode (d) optic cup (f) Retina Lens vesicle (e) Lens Mature eye Retina 36  THE ANATOMY OF DEVELOPING NERVOUS SYSTEMS 2.5 Secondary neurulation in vertebrates While primary neurulation generates much of the neural tube of higher vertebrates (Fig. 2.9(a)), the lumen of its caudal part is formed by the hollowing out of an initially solid rod of cells rather than the rolling of the neural plate. This is called secondary neurulation (Fig. 2.9(b)). Most Vertebrates Some fish Notochord (c) Tail bud Posterior Anterior Endoderm (a) PRIMARY NEURULATION (b) SECONDARY NEURULATION Neural Neural plate keel Folding Neural rod Neural rod Neural tube Cavitation Cavitation Fig. 2.9 Variations in neurulation at different positions along the neural tube and in different species. (a) Primary neurulation, involving the rolling or folding of the neuroectoderm around a central lumen, occurs along much of the length of the neural plate in most vertebrate species. (b) In posterior regions, secondary neurulation involves the initial formation of a rod of cells which then cavitates. (c) In some species of fish such as zebrafish, the neural tube forms by a thickening of the neural plate into a so-called neural keel due to movement of neuroectodermal cells towards the midline (arrows); the keel becomes a rod of cells that cavitates.  FORMATION OF INVERTEBRATE AND VERTEBRATE PERIPHERAL NERVOUS SYSTEMS  37 Secondary neurulation occurs as the vertebrate body elongates along its rostral to caudal axis. A structure called the tail bud forms at its cau- dal end (Fig. 2.9). Cells from the caudal end of the neuroectoderm gen- erate a solid caudally located rod that cavitates to form the caudal neural tube. Interestingly, they also generate mesodermal cells that contribute to the tail bud, notochord and somites; it is possible that caudal neuroectodermal and mesodermal cells originate from cau- dally-located stem cells that have the ability to generate several differ- stem cell a relatively unspecialized ent types of differentiated cell (i.e. are multipotent). The outcome of cell that can divide repeatedly to re- secondary neurulation is the generation of the caudal neural tube generate itself (self-renewal) and give which is continuous with neural tube generated by primary neurula- rise to more specialized cells, such as tion. Secondary neurulation resembles the process that generates the neurons or glia. entire neural tube in some species of fish. In these animals, the first step in neurulation generates a neuroectodermal structure called the neural keel, which is a thickening of the neural plate along its rostral to caudal axis Fig. 2.9(c). Subsequent steps result in the formation of a solid rod of cells that cavitates along its length. 2.6 Formation of invertebrate and vertebrate peripheral nervous systems The descriptions above have concentrated largely on the development of central nervous systems. Peripheral nervous systems are made up from all the neurons that reside or extend axons outside the central nervous system. They are essential for extracting sensory information from the animal’s internal and external environment and for control- ling the actions of the body. 2.6.1 Invertebrates The sensory nervous system of Drosophila pro- Shaft cell vides an excellent model for the study of mechanisms regulating the development of specific cell types. It is represented largely by Bristle small sense organs called sensilla (singular: shaft sensillum); a diagram of one is on the right. These comprise one or more bipolar neurons and a number of support cells. Socket There are different types of sensillum for dif- ferent sensory modalities. The most visible type is the sensory bristle, which consists of Sheath cell one sensory neuron and three support cells Bipolar sensory neuron that form the bristle shaft and socket, and a sheath cell for the neuron (shown on the right, taken from Fig. 2.2 earlier in this chapter). Sensilla develop from individual ectodermal cells called sense organ precursors (SOPs) (also known as sensory mother cells). Their locations and development were illustrated earlier, on the left- bipolar neuron a neuron with two hand side of Fig. 2.2(b)–(d). SOPs appear in a segmentally repeated projections emanating from the cell pattern just like neuroblasts, except that they are derived mostly body or soma. 38  THE ANATOMY OF DEVELOPING NERVOUS SYSTEMS from the lateral ectoderm. Each SOP then divides in a highly stereo- typic manner to give the four cells that differentiate to give the sen- sillum. Depending on the location of the SOP, a fifth cell from this multipolar neuron a type of neuron lineage either dies, forms a glial cell, or forms a separate multipolar that has a single axon and several neuron. At the end of embryogenesis, there are just 43 sensory neu- dendrites extending from its body. rons on each side of each larval body segment, although there are many more in the head. The adult is also covered with sensilla (flies are notably bristly), but these arise much later in the pupa during metamorphosis. Within the pupa are epithelial sheets of undifferentiated ectodermal cells called imaginal discs, which give rise to the external cuticle of the fly. SOPs for adult sensilla are formed in these imaginal discs in pat- terns that are highly stereotypic. This feature of the larval and adult PNS has made them extremely useful in uncovering basic genetic mechanisms of neural development; this work will be discussed fur- ther in Chapters 4 and 5. 2.6.2 Vertebrates: the neural crest and the placodes In vertebrates, most cells of the peripheral nervous system come from the neural crest (Figs 2.4(c), 2.6(d) and 2.10), which originates from Midbrain Neural crest (a) (b) Forebrain Pigment cells Dorsal root ganglia neurons Hindbrain Sympathetic ganglia Limb bud Adrenal medulla Aortic plexuses Enteric Notochord nervous system Aorta Gut Fig. 2.10 Diagram illustrating the paths taken by neural crest cells in the trunk of a chick embryo. (a) The embryo seen from the side is cut as shown by the line to give (b) a cross section through the trunk. Some neural crest cells migrate under the skin and form melanocytes (pigment cells); others migrate in more ventral directions to regions alongside the neural tube (where they form dorsal root ganglia), near to the aorta (where they form autonomic neurons, aortic plexuses and adrenal medulla) and around the gut (where they form the enteric nervous system).  FORMATION OF INVERTEBRATE AND VERTEBRATE PERIPHERAL NERVOUS SYSTEMS  39 the lateral edges of the neuroectoderm. Once these edges have joined autonomic neurons neurons in the to form the neural tube, neural crest cells end up dorsally along the peripheral nervous system that control line of fusion (Fig. 2.4(c)). Neural crest cells migrate widely throughout body functions below the level of con- the body, contributing sensory neurons, autonomic neurons, neu- sciousness, such as respiration, heart rons that innervate internal organs such as the gut, Schwann cells rate, digestion, and so on. and other peripheral glial cells (Fig. 2.10). The neural crest also gener- Schwann cells glial cells of the pe- ates non-neural cells. Some of these contribute to structures including ripheral nervous system that produce the adrenal medulla; others are pigment cells (called melanocytes) that the myelin sheath around axons. migrate to the skin, hair and eyes. Rostrally, neural crest cells from cra- nial regions contribute to non-neural head structures including bone, cartilage and connective tissue. Throughout the body, peripheral neurons and glia gather into dense collections called ganglia (Fig. 2.10). Ganglia in the head of vertebrates, such as the trigeminal ganglion (which transmits sensa- tion from the face), the vestibulo-cochlear ganglion (which trans- mits signals from the ear) and others, are derived not only from neural crest but also from bilateral thickenings of the ectoderm of the vertebrate head, called cranial placodes (Fig. 2.11). As well as contributing neurons to sensory cranial ganglia, the placodes gener- ate sensory structures including the olfactory sensory epithelium, lens and inner ear. Geniculate Trigeminal Otic Forebrain Olfactory Petrosal Lens Nodose Lens Trigeminal Olfactory Geniculate Petrosal Otic Somites Nodose Fig. 2.11 The cranial placodes, shown on a diagram of a chick embryo. Similar to SOPs in Drosophila (Fig. 2.2 and Section 2.6.1), the cranial placodes arise in lateral ectoderm. Most rostral are the olfactory placodes, which generate the olfactory epithe- lium and its sensory neurons, and the lens placodes. Further caudally lie the trigeminal, geniculate, petrosal and nodose placodes, all of which contribute neurons to cranial sensory ganglia, and the otic placode, which generates the inner ear including its sen- sory epithelium and the auditory and vestibular ganglia. 40  THE ANATOMY OF DEVELOPING NERVOUS SYSTEMS 2.6.3 Vertebrates: development of sense organs The way in which the lens develops from the lens placode has already been described in Box 2.2. The development of the inner ear provides another good example of how vertebrate sense organs develop. The vertebrate inner ear is made up of a labyrinth of fluid-filled chambers. These are derived from the otic placode, whose position alongside the developing hindbrain is shown in Fig. 2.11. The otic placode invaginates (a bit like the lens vesicle, Box 2.2) and pinches off from the surface ectoderm to form the otic vesicle (or otocyst) (Fig. 2.12). As the otic vesicle grows its shape changes dramatically, generating the complex structure of the inner ear (Fig. 2.12). Spe- cific parts of the otic vesicle give rise to (i) the hair cells that detect the sensory stimuli and (ii) the auditory sensory neurons that Eardrum Semicircular (a) canal Auditory External ear nerve to brain Cochlea (b) (c) (d) (e) (f) Otic vesicle Otic placode Developing ganglion of the auditory nerve Fig. 2.12 (a) The ear in humans showing its major components, including the vestibulo-cochlear nerve (also known as the audi- tory or eighth cranial nerve) responsible for transmitting information on sound and balance to the brain. (b) to (f) Steps in the early formation of the inner ear studied in chick, from the otic placode through the formation of a cup and eventually a fluid-filled chamber with the vestibulo-cochlear ganglion of the auditory nerve developing on its medial side.  SUMMARY  41 innervate them. The molecular mechanisms which control inner ear hair cell formation will be discussed in Chapter 5.4 2.7 Summary  The early embryos of animals are organized into three primary tis- sues called the germ layers: an outer layer called the ectoderm, a middle layer called the mesoderm and an inner layer called the endoderm. Ectodermal derivatives include the epidermis (or skin) and the nervous system, which is derived from the neuroectoderm.  There is great variation from species to species in the shapes of the three germ layers but their presence and relative positions remain conserved.  Formation of the germ layers occurs at gastrulation. Gastrulation involves the movement of cells from the outer surface of the embryo to its inside.  The mature central nervous system of vertebrates is hollow. In most species this reflects its origin from a hollow structure in the embryo known as the neural tube, which is formed by the folding of an earlier structure called the neural plate.  The anterior part of the neural tube forms the forebrain, the mid- brain and the hindbrain. The posterior part of the neural tube forms the spinal cord.  Most cellular proliferation occurs on the inner side of the neural tube closest to its lumen. Cells destined to become neurons migrate away from this proliferative zone towards the outside of the tube to differentiate into mature neurons.  Cells along the dorsal neural tube give rise to the neural crest.  The peripheral nervous systems of flies develop from ectodermal cells called sense organ precursors (SOPs). In vertebrates, most cells of the peripheral nervous system come from the neural crest. Others are derived from bilateral thickenings of the ectoderm of the verte- brate head, called cranial placodes. The placodes generate sensory structures including the olfactory sensory epithelium, lens and inner ear. 4 For more on this topic, see Ladher, R. K., O’Neill, P. and Begbie, J. (2010) From shared lineage to distinct functions: the development of the inner ear and epibranchial placodes. Development, 137, 1777– 85.