Molecular control of brain development Neuronal Patterning and Regionalization

2012

The Spemann Organizer

In 1924, the Ph.D. student Hilde Mangold working in the laboratory of German embryologist Hans Spemann performed an experiment that demonstrated that the pattern of development of cells is influenced by the activities of other cells Spemann and Mangold knew that the cells that develop in the region of the gray crescent migrate into the embryo during gastrulation and form the notochord (the future backbone; made of mesoderm). She cut out a piece of tissue from the gray crescent region of one newt gastrula and transplanted it into the ventral side of a second newt gastrula. To make it easier to follow the fate of the transplant, she used the embryo of one variety of newt as the donor and a second variety as the recipient.

The results: the transplanted tissue developed into a second notochord neural folds developed above the extra notochord ,these went on to form a second central nervous system (portions of brain and spinal cord) and eventually a twoheaded tadpole. But the most remarkable finding of all was that the neural folds were built from recipient cells, not donor cells. In other words, the transplant had altered the fate of the overlying cells (which normally would have ended up forming skin [epidermis] on the side of the animal so that they produced a second head instead! Spemann and Mangold used the term induction for the ability of one group of cells to influence the fate of another. And because of the remarkable inductive power of the gray crescent cells, they called this region the organizer.

Organizer Transplant experiment

A region just above the blastopore lip (mesodermal tissue) is excised & transplanted to ventral side of host. The host embryo develops a secondary dorsal axis, first evident by a secondary neural plate.

A section through a host embryo with two dorsal axes: Secondary dorsal axis contains the same tissues as the primary dorsal axis, including a nervous system.

As neural tissue was derived from recipient cells, not

donor cells the transplant had altered the fate of the overlying cells

The Organizer of Spemann and Mangold.

Dorsalization of mesoderm and neural induction by Spemanns Organizer during The Organizer experiment (Spemann and Mangold, 1924) is the best known experiment in embryology. It has led to the current view that development occurs through a cascade of cell-cell interactions. If the dorsal lip (the site where gastrulation starts) of the blastopore is transplanted to the opposite side of the embryo, it is able to recruit host cells organizing them into a secondary (twinned) body axis containing many histotypes and complex structures. Spemann referred to the dorsal lip as a primary organizer.

Restriction of Cellular Potency. The fate of embryonic cells is affected by both the distribution of cytoplasmic determinants and by cleavage pattern.

Steps during neural development

Neurogenesis Compartmentalization Neural differentiation Neural migration Axonal guidance Synaptogenesis

Neural development in vertebrate embryo: Gastrulation

Blastula stage embryo with 3 germ layers, first signs of invagination of dorsal blastopore lip

Embryo in midgastrulation, involution of dorsal mesoderm (organizer tissue). Gastrula stage embryo: Embryo at end of gastrulation. The 3 germ layers have arrived at their final destination

Blastula stage through neurulae, highlighting gastrulation and neurulation.

Organizing Centers:
Restricted specialized areas that are crucial for the induction of area specification

Spemanns organizer (dorsoblastopore lip) Hensens node (similar to Spemanns org) Roofplate and notochord become organizers secondary organizers:
Isthmic organizer (IsO) Anterior neural ridge (ANR) Cortical hem

Default model of neural induction . Balance between agonists and

antagonists! Importance of inhibition as a developmental regulatory mechanism

Expression of signaling factors:

Bone morphogenic protein (BMP), a TGF--like polypeptide growth factor (PGF )expressed in ectoderm on ventral side, inducing ectoderm to become epidermis.

Organizer on the dorsal side releases inhibitors of the BMPs: noggin, chordin, and follistatin, which diffuse into the ectoderm on the dorsal side, block the effects of BMPs, and allow neural tissue to form.

Signaling pathway involving BMPs

Large family of polypeptide growth factors (PGF) related to transforming growth factor- (TGF-): BMP, activin, and GDF group members. Heterodimer receptors, with type I & type II subunits, cytoplasmic domains with serine/theronine kinase activity. Transforming growth factor beta (TGF-beta) and activin bind to receptor complexes that contain two distantly related transmembrane serine/threonine kinases known as receptor types I and II. The type II receptors determine ligand binding specificity, and each interacts with a distinct repertoire of type I receptors. Dimerization after binding of a TGF--like PGF starts signal transduction pathway: Activation of cytoplasmic proteins (SMADs), which translocate to nucleus to activate expression of downstream target genes. Inhibitory mechanisms regulate signaling: Extracellular proteins such as chordin, tolloid, and twisted gastrulation interact with the BMP-like ligands, regulating their diffusion through the extracellular milieu and their ability to bind receptor Cell surface proteins such as BAMBI inhibit signaling by binding up BMPs but failing to transduce a signal. Inhibitory SMADs poison the signal transduction pathway.

Signaling transduction pathway

involving BMPs
1.Activation of cytoplasmic proteins (SMADs), which translocate to nucleus to activate expression of downstream target genes. 2.Inhibitory mechanisms regulate signaling:
Extracellular proteins such as chordin, tolloid, and twisted gastrulation interact with the BMP-like ligands, regulating their diffusion through the extracellular milieu and their ability to bind receptor Cell surface proteins such as BAMBI inhibit signaling by binding up BMPs but failing to transduce a signal. Inhibitory SMADs poison the signal transduction pathway.

Neurulation
The neural plate forms after gastrulation is completed. The neural tube narrows along its medial-lateral Axis. The plate begins to role into a tube. The cells at the midline produce a medial hinge point (MHP). As the tube forms and segregates into the embryo, neural crest cells emigrate from the dorsal aspect of the neural tube.

Steps during neural development

Neurogenesis Compartmentalization Neural differentiation Neural migration Axonal guidance Synaptogenesis

Early Neural Patterning: Establishment of AP Axis

In the early stages of pattern formation, two perpendicular axes are established -Anterior/posterior (A/P, head-to-tail) axis -Dorsal/ventral (D/V, back-to-front) axis Polarity refers to the acquisition of axial differences in developing structures Position information leads to changes in gene activity, and thus cells adopt a fate appropriate for their location
15

AP polarity of vertebrate CNS

Head organizer becomes precordal mesoderm (PME) underneath prechordal plate Tail organizer becomes notochord and somites, underneath epichordal neural plate Head and tail organizer release factors which create a gradient.

Neural Patterning
A/P polarity and other key organizational features are first established by gradients of positional information of a gradient of a substance or signal. Gradient confer positional information as the relative concentrations correlated with distance. In the 3-dimensional system of the embryo, the initial establishment of A/P polarity is signalled by the organizer (dorsal lip of the blastopore in amphibians; Hensens node in birds). During gastrulation, the organizer tissues come to underlie the neural plate and differentiate into the notochord. The chordal mesoderm, which underlies the future midbrain, hindbrain, and spinal cord, apparently sends out distance signals from prechordal mesoderm.

The candidate neural inducers, (chordin, noggin, and follistatin) induce primitive neural tissue that appears to be forebrain-like; chordin particularly potent. These 3 proteins antagonize members of TGF- signalling family of molecules. This suggests that induction of anterior neural plate differentiation involves inhibitors of TGF--like signals that repress neural development. This would be a ground state, which would be induced to be more posterior by a 2nd signal: a transforming signal. In this case, a type of gradient, a ratio between activating (noggin) and transforming signals would determine the A/P polarity along the neuraxis. Possible candidate posteriorizers (transforming signals) include bFGF and retinoic acid.

Tail organizer: Head organizer: BMP Inhibitors Cordin and Noggin, Wnt inhibitors Cerberus, Dickkopf and frzb1 to "anteriorize" neural tube FGF, WNT, RA &BMP inhibitors are posteriorizing signaling molecules

The neural tube, shown here for a mouse, is subdivided into four longitudinal domains: the floor plate, basal plate, alar plate, and roof plate. Motor neurons are derived from the basal plate.

Dorsal Ventral pattern: Notochord as organizer

Experiment using Pax gene-Left: During development, the floor plate (red) develops above the mesodermal notochord (n) and motor neurons (yellow) differentiate in adjacent ventrolateral region of the neural tube. Center: Grafting a donor notochord (n') alongside the folding neural plate results in formation of an additional floor plate and a third column of motor neurons. Right: Removing the notochord from beneath the neural plate results in the permanent absence of both floor plate and motor neurons in the region of the extirpation. Pax6 expression (blue) extends through the ventral region of the cord.

Floor plate cells are induced by sonic hedgehog (SHH) secreted from the notochord whereas ventral midline cells of the rostral diencephalon (RDVM cells) appear to be induced by the dual actions of SHH and bone morphogenetic protein 7 (BMP7) from prechordal mesoderm.

Sonic-hedge-hog expression by notochord & floor plate, control of ventral patterns

Shh activity in the ventral neural tube (blue dots) is distributed in a ventral-high, dorsal-low profile within the ventral neural epithelium. 5 classes of neurons are generated in response to graded Shh signalling
T.M. Jessell, 2000

Sonic Hedge hog gene and neuron

Shh acts as a morphogen, forming a gradient in the ventral neural tube, to which cells differentiate in a concentration- dependent fashion SHH is a member of the hedgehog family of signalling molecules identified by homology to the Drosophila hedgehog (HH). SHH is proteolytically cleaved to produce two secreted proteins , a 19 kDa N-terminal protein (N-SHH) that mediates all signalling activities in vertebrates and invertebrates and a 25 kDa C-terminal protein (C-SHH) that possesses protease activity. N-SHH is responsible for a number of early patterning processes; it is involved in the control of leftright asymmetry, dorsoventral patterning of the CNS and somites, patterning of the limb, as well as in some aspects of organogenesis Sonic hedgehog is a secreted extracellular protein that transmits its signal by binding to a receptor on the surface of a cell. That binding, in turn, propagates the signal to the interior of the cell. Once inside, the signal activates a variety of genes that begin to change a generic neuron into a motor neuron. Signalling by a SHH gradient establishes distinct progenitor domains by regulating the expression of a set of homeodomain proteins that comprises members of the Pax, Nkx, Dbx and Irx families

(a) Schematic representation of the neural tube and underlying axial mesoderm with regions expressing Shh indicated in red. Line shows position of transverse section shown in (b). T, telencephalon; D, diencephalon; M, midbrain; H, hindbrain; S.Cord, spinal cord; PM, prechordal mesoderm; NC, notochord.

(b) Transverse section at the level of the spinal cord, showing expression of Shh in the notochord and floor plate.

(c) Cell types induced by Shh vary according to their position along the anteroposterior axis. Different colours indicate regional differences in the cell types differentiating in response to Shh signalling. Dark blue, ganglionic eminence; pale blue, RDVM cells; brown, dopaminergic neurons; yellow, serotonergic neurons; green, motor neurons.

(d) Transverse section at level of spinal cord (indicated in panel c) showing ventrolateral cell types (green) arranged with bilateral symmetry around ventral midline floor plate cells (red).

(a) Distinct ventral cell types differentiate at stereotyped positions in the ventral neural tube. FP, floor plate; MN, motor neurons; V0V3, classes of ventral interneurons generated at spinal cord levels.

(b) Proposed gradient of Shh signal moving from its sources of expression in the ventral neural tube and notochord

(c) The concentration of Shh required to induce specific ventral cell types in vitro correlates directly with their dorso-ventral position in vivo.

Patterning along the dorso-ventral axis: a graded Shh signal

Gradient model for the induction of ventral cell types by Shh.

SHH signalling pathway

At the cell surface, SHH binds with high affinity to patched (Ptc), a 12- transmembrane protein. In mammals, two isoforms of Ptc are encoded by Ptc1 and Ptc2, although Ptc1 appears to be active in the CNS . Binding of SHH to Ptc prevents the normal inhibition of smoothened (Smo), a seven-transmembrane protein with a topology reminiscent of G-protein-coupled receptors, which is the signalling component of the SHH-receptor complex. During development of the vertebrate CNS, either inhibition of Gi proteins or expression of a constitutively active form of Smo is sufficient to trigger some actions of SHH.

SHH signalling pathway

Hedgehog-interacting protein (Hip) is a type I transmembrane protein that attenuates SHH signalling by binding N-SHH with an affinity similar to that of Ptc1 Vitronectin, an extracellular matrix glycoprotein, enhances SHH activity during motor-neuron differentiation, also by binding SHH directly. Within the nucleus of the responding cell, zinc-finger transcription factors of the Ci/GLI family (GLI13) act at the last known step of the SHH-signal-transduction pathway , although it is still unclear whether GLI proteins mediate all aspects of SHH signalling during vertebrate CNS development .

Binding of SHH to Ptc prevents the normal inhibition of smoothened (Smo), During development of the vertebrate CNS, either inhibition of Gi proteins or expression of a constitutively active form of Smo is sufficient to trigger some actions of SHH. Within the nucleus of the responding cell, zinc-finger transcription factors of the Ci/GLI family (GLI13) act at the last known step of the SHH-signaltransduction pathway

(a) Progenitor domains corresponding to the differentiation of specific ventral cell types (a) are shown on the left-hand side and indicated with a letter P. Each domain can be recognised by the combinatorial pattern of gene expression shown on the right.

(b) Shh initiates the specification of progenitor cell domains by first exerting a graded repression of a number of genes which would otherwise be expressed more widely in the neural tube. These genes include Pax6, Irx3 and members of the Dbx family

(c) The repression of Pax6 by Shh may indirectly allow the expression of Nkx2.2 in a discrete domain adjacent to the floor plate (P3). A reciprocal repression between these two genes may then act to refine and maintain the boundary between the P3 and PMN domains. Similar mechanisms are believed to occur at the boundaries between other ventral progenitor domains.

Establishment and maintenance of progenitor cell domains in the ventral neural tube

Model for ventral neural patterning by SHH.

Left: Graded SHH signaling from the ventral pole induces expression of some homeobox genes (e.g., Nkx2.2, Nkx6.1) and represses existing expression of others (e.g. Pax6, Dbx2).

Center: Cross-repressive interactions

between pairs of transcription factors sharpen mutually exclusive expression domains.

Right: Profiles of homeobox gene expression define progenitor zones and control neuronal fate.

Regulation of DV pattern in the telencephalon by SHH.

Cross section of mouse telencephalon stage. SHH produced in the ventral midline region controls development of basal ganglia primordia and medial and lateral ganglionic eminences (MGE, LGE). First, ventral SHH induces medial ganglionic eminences (MGE) gene expression; SHH (partly produced by the MGE) induces lateral ganglionic eminences (LGE )gene expression later.

Two Critical Periods of Sonic Hedgehog Signaling Required for the Specification of Motor Neuron Identity
SHH activity is required for the induction of floor plate differentiation by the notochord and independently for the induction of motor neurons by both the notochord and midline neural cells. Motor neuron generation depends on two critical periods of SHH signaling: 1. an early period during which naive neural plate cells are converted into ventralized progenitors 2. a late period that extends well into S phase of the final progenitor cell division, during which SHH drives the differentiation of ventralized progenitors into motor neurons. The ambient SHH concentration during the late period determines whether ventralized progenitors differentiate into motor neurons or interneurons, thus defining the pattern of neuronal cell types generated in the neural tube.

Dorsoventral axis of the spinal cord in quail (bird)

Classes of neurons that can be identified along the dorsoventral (DV) axis of the normal embryonic spinal cord. D, dorsal sensory neurons; fp, floor plate; mn, motor neurons; rp, roof plate; V0, V1, V2, interneurons; V3, ventral neurons. These classes of neurons are distinguished by their unique gene-expression profiles, many of which are characterized by combinations of homeobox transcription factors.

b | Dorsal patterning is controlled by a gradient of bone morphogenetic proteins (BMPs) that arises from the dorsal roof plate, and ventral patterning is controlled by a gradient of sonic hedgehog (Shh) that arises from the floor plate.

d| The role of RA in generating a subset of motor neurons in the spinal cord. Retinaldehyde
dehydrogenase 2 (Raldh2) is expressed in motor neurons at limb levels (red circles). A subset of motor neurons known as lateral motor column neurons (LMCs) originates close to the midline of the cord (green circles) and then migrates through the Raldh2expressing motor neurons to differentiate at the edge of the cord (arrow). During this journey, these cells are exposed to RA released by the motor neurons (red circles), and as a result, are induced to form LMCs.

c | The pattern of dorsal and ventral genes in the retinoic acid (RA)-depleted quail spinal cord indicates that there is increased ventral signalling and decreased dorsal signalling.

VERTEBRATE CNS DEVELOPMENT

Early neural tube with dorsoventrality : At the dorsal pole is the roof plate (rp), a single line of cells with the nuclei at the margin, and at the ventral pole is the floor plate (fp) where the cells are similarly arranged. In the body of the neural tube, there are many densely packed neuroblasts, but towards the ventral region, there is a swelling where the neuroblasts are not so densely packed and these are the presumptive motor neurons (mn).
(B) The neural tube in A differentiates into many different neuronal types, the major ones are shown here. Sensory neurons from the dorsal root ganglia (drg, purple) enter the dorsal cord and synapse there. In the ventral region, the motor neurons differentiate (mn, red). In between these two are various types of interneurons with axon trajectories which connect the sensory and motor regions (blue neuron) or connect one side of the cord to the other (yellow neuron).

(C) Diagram to show the regionalization of the 6 types of dorsal neurons (dl1dl6) and the 5 types of ventral neurons (v0v3 + mn) in the developing neural tube. On the left are the gene and protein markers which are used to identify the progenitors domains (in the ventricular region close to the midline) of these different DV regions. On the right are the gene and protein markers which are used to identify the neuronal types (in the mantle region where neurons differentiate)

Summary diagram of the posterior end of the embryo where DV patterning is taking place.

In the stem zone, FGFs from the underlying mesoderm (blue) prevent neural differentiation in the overlying neural plate (signal 1). In the transition zone, the notochord differentiates and starts to express Shh (yellow, signal 3). The somites differentiate and start to express Retinaldehyde dehydrogenase 2 , RALDH2 (red) which synthesizes RA (signal 2). BMPs start to be produced form the roof plate (green, signal 4). In the neuronal differentiation and DV patterning zone, RA antagonizes FGF and vice versa, RA induces a specific set of genes in the neural tube (red arrow), Shh is induced in the floor plate and spreads dorsally in a concentration gradient (yellow arrow), and BMPs spread ventrally in a concentration gradient (green arrow).

Summary of the gene interactions involved in neuronal differentiation in the neural tube. (A) Network showing the relationship between the inducers of Class I and Class II genes and how they themselves interact. (B) Later neuronal differentiation of motor neurons involves multiple use of a RA signal and multiple use of the induction of repressors

Regionalization of the Nervous System

I. Segmentation II. Developmental control genes (e.g., Hox), which encode positional values along A/P axis.A positional signaling mechanism activates these genes . E.G. In birds, At Hensons Node (similar to blastopore of higher animals), a strong candidate for this signal is a gradient of retinoic acid, which regulates the pattern of Hox gene expression. Different Hox genes at specific locations respond more or less readily to lower or higher [RA]s, through a family of receptors, which, bound by RA, become transcription factors.

I.

Segmentation: Subdivision of the main body axis by segmentation provides compartments, which allocate precursor cells into a repeated set of similar molecules, so that developmental fields can remain small, and specialization of cell types and patterns can be generated as local variations on the repetitive theme. e.g. Mesoderm = segmented into somites, yielding muscle groups. The neuraxis is also segmented In the CNS, segmentation is a mechanism for specifying pattern during development. The earliest neurons and neural pathways are laid out in stripes, which match a morphological repeat pattern ( a 2-segment repeat pattern, which has similar patterns of development in even- or odd-numbered segments).

Cells are segregated by: a. Mechanical boundaries (certain extracellular matrix pattern, such as chondroitin SO4 appear at the boundaries during development (however, only important during later level.). b. Differential adhesion between cells (occurs through a 2-segment repeat rule (evens evens; odds odds), so that adjacent rhombomeres remain separate.

Compartmental organization of hindbrain into rhombomeres in Zebra fish

(a) Genes expressed in restricted domains (represented in red and blue) within the anteroposterior axis of the hindbrain initially show diffuse boundaries. For example, krox20 expression (shown on the right as blue signal following in situ hybridization) shows diffuse boundaries in presumptive r3 and r5 at bud stage (10 hours postfertilization),

Genes are expressed in alternate stripes that correspond with presumptive Rhombomeres.

(b) Gene expression domain boundaries progressively sharpen to form straight interfaces. At 18 somites (18 hours post-fertilization), domains of krox20 expression are sharply restricted in presumptive r3 and r5. (c) Gene expression domain boundaries coincide with structural boundaries; actin accumulation (shown on the right as red signal after alexa-redphalloidin staining) transiently delineates rhombomere boundaries (white arrowheads) (d) Mature rhombomere boundary zones are characterized by large intercellular spaces (white dots) and concentrations of axons. Different types of cell differentiate at stereotypical positions with respect to the boundary (indicated by gradient of shading across each rhombomere). Expression of mariposa (shown on the right as blue signal following in situ hybridization) is localized to rhombomere boundary zones.

Restriction of movement of mitotic Precursor cells across interfaces.

Julie E. Cooke, Cecilia B. Moens, 2002: Schematic dorsal views of part of the developing vertebrate hindbrain (left side of each panel) and dorsal views of flat-mounted zebrafish embryo hindbrains at corresponding stages (right side of each panel). Anterior is to the left; scale bars, 50 m

The interfaces between Rhombomeres acquire molecular and Morphological specialization marked by distinct boundaries.

Mechanism for Hindbrain Segmentation. Hindbrain segmentation and the generation of sharp domains of gene expression is a two step process. Initial gene expression boundaries in the hindbrain are diffuse and bidirectional repulsive signaling mediated the Eph/ephrin gene families leads to a sorting of cells based on appropriate gene expression. Concomitant with the morphological formation of rhombomere boundaries, cells isolated on the wrong side of the border exhibit plasticity in their gene expression patterns in response to cell community signaling effects. Together this leads to the formation of the sharp molecular and cellular boundaries that are characteristic of vertebrate hindbrain development.

Stages in the compartmental organization of rhombomeres

Genes such as Krox20 and EphA4 (blue) and ephrin-B2 (pink) are expressed in alternate, fuzzyedged stripes (left). Subsequently, restriction to the movement of mitotic precursor cells occurs at the interfaces between newly formed rhombomeres, which are now sharply defined, and marked by increased intercellular spaces. (right)

Sharpening of boundaries and cell lineage restriction occur through the interaction of Eph and ephrin molecules.

Neurons and synapse

Once a neuron acquires its individual identity and stops dividing, it extends its axon with an enlarged tip known as a growth cone. The growth cone is specialized for moving through tissue, using its skills to select a favourable path. As it does so, it plays out the axon behind it. Once its target has been reached the growth cone loses its power of movement and forms a synapse. Axonal guidance is a supreme navigational feat, accurate over short and long distances. It is also a very single-minded process for not only is the target cell selected with high precision but, to get there, the growth cone may have to cross over other growth cones heading for different places. Along the path, guidance cues that attract (+) or repel (-) the growth cones help them find their way, although the molecular mechanisms responsible for regulating the expression of these cues remain poorly understood.

Pattern Generation does not Involve only the Migration of Cells themselves, but also the Axons of Cells Extension or travel of a neuronal axon to a given area and making specific connections Appears to involve three steps:
pathway selection axons travel to specific region of embryo target selection recognize and bind a set of cells address selection refine binding to one or a subset of initial target (first two dont depend on neuronal activity)
Role of the substrate in directing the pathway of axons has been experimentally shown as neuronal growth cones prefer to migrate over adhesive surfaces coated with laminin Some substrates cause repulsion of axons e.g. ephrin or semiphorin proteins. But all axons may not be repulsed by these molecules. Some may be attracted.

The structure of the growth cone is fundamental to its function. The leading edge consists of dynamic, finger-like filopodia that explore the road ahead, separated by sheets of membrane between the filopodia called lamellipodia-like veils (see the figure). The cytoskeletal elements in the growth cone underlie its shape, and the growth cone can be separated into three domains based on cytoskeletal distribution. The peripheral (P) domain contains long, bundled actin filaments (F-actin bundles), which form the filopodia, as well as mesh-like branched F-actin networks, which give structure to lamellipodia-like veils. Additionally, individual dynamic 'pioneer' microtubules (MTs) explore this region, usually along F-actin bundles. The central (C) domain encloses stable, bundled MTs that enter the growth cone from the axon shaft, in addition to numerous organelles, vesicles and central actin bundles. Finally, the transition (T) zone sits at the interface between the P and C domains, where actomyosin contractile structures (termed actin arcs) lie perpendicular to F-actin bundles and form a hemicircumferential ring. The dynamics of these cytoskeletal components determine growth cone shape and movement on its journey during development.

Axon growth, requires (A) the supply of building blocks as well as (B) cycling filaments in the growth cone (C) connections between growth cone filaments and the growth substrate

Goldberg J L Genes Dev. 2003;17:941-958

2003 by Cold Spring Harbor Laboratory Press

The growth cone-Extracellular guidance molecules bind surface receptors on the growth cone.

In turn, these receptors activate signalling cascades that ultimately influence growth cone cytoskeletal components controlling morphology and motility. Signalling cascades are known to affect the actin cytoskeleton. It is not clear whether there are also direct influences on growth cone microtubule assembly during axon guidance.
.

Oster S F , Sretavan D W Br J Ophthalmol 2003;87:639-645

2003 by BMJ Publishing Group Ltd.

Synapse Generation
e.g. formation of a synapse at a neuromuscular junction

a)growth cone approaches muscle cell b)axon forms unspecified contact on cell surface. Agrin from neuron causes clustering of Ach receptors c)neurotransmitter vescicles enter terminal and extracellular matrix connects the two d)other axons converge f)all but one axon eliminated, axon branches, folds form in muscle cell membrane, Schwann cell covers axon.

Genetic Control of Dendrite Development

Dendrite arborization patterns are critical determinants of neural circuit formation and influence the type of synaptic or sensory inputs a neuron is able to receive. Relatively little is known about the molecular mechanisms that control dendrite development. 1. Regulation of dendritic field size and complexity by transcription factors: In some cases, the "dendritic fate" of a particular neuron might be specified by a single transcription factor. For example, in the Drosophila PNS, the zinc fingercontaining protein Hamlet functions as a binary switch between the elaborate multiple-dendrite morphology of the da neuron and the single, unbranched dendrite morphology of the external sensory (es) neuron. In most cases, however, the dendritic fate is determined by the combined action of multiple transcription factors.

Genetic Control of Dendrite Development in Drosophila

Drosophila da (dendritic arborization) neurons fall into four distinct morphological classes (IIV). The selector gene Cut is expressed at different levels in the da neurons. Neurons with small and simple dendritic arbors either do not express Cut (class I neurons) or express low levels of Cut (class II). Neurons with more complex dendritic branching patterns and larger dendritic fields (classes III and IV) express higher levels of Cut. Cut levels are a critical determinant of da neuron class-specific dendritic morphologies. In contrast to Cut, Spineless (Ss), the homolog of the mammalian dioxin receptor, is expressed at similar levels in all da neurons. Studies of the epistatic relationship between Cut and Ss indicate that these transcription factors are likely acting in independent pathways to regulate morphogenesis of da neuron dendrites. More than 70 transcription factors regulate dendritic arbor development of class I neurons in fly, suggest that complicated networks of transcriptional regulators likely regulate type-specific dendrite arborization patterns.

Genetic Control of Dendrite Development in Drosophila

2. The molecular mechanism for dendritic self-avoidance and tiling. The dendrites of each da neuron show self-avoidance and tend to spread out without crossing over. Class III and class IV da neurons show tiling; i.e., there is little overlap between the dendritic fields of adjacent neurons of the same class because their dendrites show homotypic repulsion. Dscam (Down syndrome cell adhesion molecule), is needed for self-avoidance and contribute to the spreading of dendrites. Without Dscam, the dendrites of each da neuron bundle together or cross over. For the dendritic fields of different neurons to coexist in the same space, they need to express different Dscam isoforms. In contrast, tiling requires some cell surface recognition molecules other than Dscam to mediate the homotypic repulsion. The evolutionarily conserved protein kinase Tricornered (Trc) and the putative adapter protein Furry (Fry) have been identified as important components of the intracellular signaling cascade involved in tiling. 3. Dendrite-specific developmental regulators are a group of dar (dendritic arborization reduction) genes. Mutations of any of the dar genes lead to defective dendritic arbors but normal axonal projections. There may be a total of about 20 dar genes in Drosophila.

Genetic Control of Dendrite Development in Drosophila

4. The maintenance of dendritic fields by specific mechanisms.The tumor-suppressor Warts (Wts), one of two NDR (nuclear Dbf2-related) family kinases in Drosophila (the other being Trc), and the Polycomb group of genes are required for the maintenance of class IV da dendrites. Loss-of-function mutants of any of those genes cause a progressive defect in the maintenance of dendritic tiling, resulting in large gaps in the receptive field 5. The remodeling of dendritic fields. Drosophila class IV da neurons undergo dramatic remodeling during metamorphosis. Early in the pupal stage, those neurons prune all their dendrites. Later each neuron grows a completely new dendrite for adult function. While the dendrites are being remodeled, the axons stay largely intact.

Extension of Drosophila Work to Dendrite Development of Mammalian Central Neurons:The great majority of the genes found to affect Drosophila dendrite development have a mammalian homolog(s). In several cases those homologs (for example, Dasm1, Dar3/Sar1) have similar function in regulating dendrite development in the mammalian central nervous system.

II. Developmental Control Genes. These genes, which encode txn factors, or signaling molecules, are expressed in a spatially variable manner. The Hox genes (homeobox family) have a clustered chromosomal organization. The relative position of the gene reflects the expression along the A/P axis. This expression of the Hox gene confers positional value and regional identity. The Hox genes are a set of transcription factor genes that exhibit an unusual property: These are genes that specify segment identity whether a segment of the embryo will form part of the head, thorax, or abdomen, for instanceand they are all clustered together in one (usually) tidy spot. Within that cluster, there is even further evidence of order. Unlike most genes, however, the order of Hox genes in the genome actually holds meaning. Hox code represents is a somewhat digital mechanism for regulating axial patterning. By mixing and matching combinations of the expression of a small number of Hox genes, organisms generate a greater range of morphological possibilities

Hox genes
E.g. Hox Genes in Drosphila :There are eight Hox genes in a row, and the genes' order within that row reflects their order of expression in the fly body. The gene found on the left or 3' end of the DNA strand, denoted lab (labial), is expressed in the head; on the other hand, the gene at the right end of the DNA strand, Abd-B (Abdominal-B), is expressed at the end of the fly's abdomen.

Knocking out individual Hox genes in Drosophila causes homeotic transformationsin other words, one body part develops into another. A famous example is the Antennapedia mutant, in which legs develop on the fly's head instead of antennae. The Hox genes are early actors in the cascade of interactions that enable the development of morphologically distinct regions in a segmented animal.e.g. the activation of a Hox gene from the 3' end is one of the earliest triggers that lead the segment to develop into part of the head.

Genomic Organization of the Hox Gene Cluster A schematic of the Hox gene clusters (not to scale) in the genomes of D. melanogaster and M. musculus. Genes are colored to differentiate between Hox family members, and genes that are orthologous between clusters and species are labeled in the same color. Genes are shown in the order in which they are found on the chromosomes but, for clarity, some non-Hox genes that are located within the clusters in the fly genome have been excluded. The positions of three non-Hox homeodomain genes, zen, bcd and ftz, are shown in the fly Hox cluster (grey boxes). Gene abbreviations: lab, labial; pb, proboscipedia; zen, zerknullt; bcd, bicoid; Dfd, Deformed; Scr, Sex combs reduced; ftz, fushi tarazu; Antp, Antennapedia; Ubx, Ultrabithorax; abd-A, abdominal-A; Abd-B, Abdominal-B.

Genes respond less rapidly; require higher [RA]s

Genes respond more rapidly at lower [RA]s

Posterior CNS

Anterior CNS

Change in Hox gene expression change in morphology along the A/P axis
The signaling mechanism for expression of these genes is a gradient of Retinoic acid (RA). The RA signal regulates the pattern of Hox expression. There is a direct correspondence between the location of the Hox gene in its cluster and its responsiveness to RA.

Patterning of the brain and spinal cord through compartmentalization:

Regional patterning: Forebrain (FB), Midbrain (MB), Hindbrain (HB) and Spinal cord (SC). Graded Wnt signaling functions along the entire length of the neuraxis inducing progressively more posterior neural fates. Hox genes play important roles in establishing regional cell identity. This is achieved via opposing gradients of RA and FGF signaling.

Regional specification in the developing brain

Three-vesicle state

Five-vesicle state

Rhombomeres the clearest subdivision partition the hindbrain neuroepithelium e.g. CHICK.

Hox gene expression domains in the CNS

Nested domains of homeotic genes along the AP axis of the Drosophila and mouse CNS. Hox genes specify a positional value along the AP axis, which is interpreted differently in fly and mouse in terms of downstream gene activation, resulting in neural structure; Hirth et al., (1998).

SUMMARY OF MOLECULAR CONTROL OF NERVOUS DEVELOPMENT

The organizer has three main properties: 1) it induces neural tissue on the overlying ectoderm, 2) imparts more dorsal characteristics to the mesoderm of the marginal zone (i.e., dorsalizes mesoderm), leading to the formation of somites and trunk muscles, 3) it induces a secondary gut (dorsalization of the endoderm).

SUMMARY

The organizer does NOT induce the central nervous system but, instead, it prevents signals originating from the ventral side of the blastula from inducing skin (epidermis) there. Cells on the ventral side of the blastula secrete a variety of proteins such as bone morphogenetic protein-4 (BMP-4) These induce the ectoderm above to become epidermis. If their action is blocked, the ectodermal cells are allowed to follow their default pathway, which is to become nerve tissue of the brain and spinal cord. The Spemann organizer blocks the action of BMP-4 by secreting molecules of the proteins chordin and noggin Both of these physically bind to BMP-4 molecules in the extracellular space and thus prevent BMP-4 from binding to receptors on the surface of the overlying ectoderm cells. This allows the ectodermal cells to follow their intrinsic path to forming neural folds and, eventually, the brain and spinal cord.

SUMMARY Organizers
ORGANIZERS IN XENOPUS Protein synthesis by the cells of the organizer requires transcription of the relevant genes (e.g., chordin). Expression of organizer genes depends first on Wnt transcription factors. Their messenger RNAs were deposited by the mother in the vegetal pole of the egg. After fertilization and formation of the gray crescent, they migrated into the gray crescent region (destined to become the organizer) where they were translated into Wnt protein. Wnt protein accumulation on the dorsal side of the embryo unleashes the activity of Nodal a member of the TGF- family. Nodal induces these dorsal cells to begin expressing the proteins of Spemann's organizer. ORGANIZERS IN DROSOPHILA Proteins similar in structure to the bone morphogenetic proteins and also to chordin are found in Drosophila. The role of BMP-4 is taken by a related protein encoded by the decapentaplegic gene (dpp). The role of chordin is taken by a related protein called SOG encoded by the gene called short gastrulation. In Drosophila, DPP is produced in the dorsal region of the embryo and SOG is produced in the ventral region.

Difference in Drosophila and Humans

The actions of SOG on overlying cells are the same as in Xenopus; that is, the SOG protein prevents the DPP protein from blocking the formation of the central nervous system. The result in Drosophila is that its central nervous system forms on the ventral side of the embryo, not on the dorsal! One of the distinguishing traits of all arthropods (insects, crustaceans, arachnids) as well as many other invertebrates, such as the annelid worms, is a ventral nerve cord. Chordates, including all vertebrates, have a dorsal (spinal) nerve cord.

Human nervous system develpment

Cell differentiation begins with the emergence of the cells in the three primordial layers of the gastrula: the ectoderm (outer layer), the mesoderm (middle layer) and the endoderm (inner layer). The progenitor cells for all the neurons and glial cells of the central nervous system begin as a further differentiation of ectoderm cells into a layer known as the neural plate. Neural plate formation is induced by chemical signals from the mesoderm (evidently peptides with molecular weight less than 1,000). The neural plate folds and differentiates into neural crest cells and a neural tube. The neural crest cells become the peripheral nervous system, whereas the neural tube becomes the central nervous system. Cells in both structures differentiate into glial cells of various types as well as into immature neurons which migrate, grow axons & dendrites, form synapses and mature.

Neurulation
The neural plate forms after gastrulation is completed. The neural tube narrows along its medial-lateral Axis. The plate begins to role into a tube. The cells at the midline produce a medial hinge point (MHP). As the tube forms and segregates into the embryo, neural crest cells emigrate from the dorsal aspect of the neural tube.

 Genes give dorsalventral position information. Sonic hedgehog is an example of a dorsalventral gene that is expressed in the notochord and induces cells in the overlying neural tube to become ventral spinal cord cells. Another family of homeobox genes, the Pax genes, are important in nervous system and somite development. Pax3 is expressed in neural tube cells that will become dorsal spinal cord cells. Pax3 and sonic hedgehog interact to determine dorsalventral differentiation of the spinal cord.

Neurulation

Neurulation. (A) During the first phase of neurulation, nave uncommitted ectoderm is induced to form neural plate tissue via BMP inhibitory signals (noggin, chordin, follistatin) secreted from the underlying mesoderm. (B) During the second phase of neurulation the two halves of the open neural plate begin to curl up to form a hollow neural tube. During this time neural crest cells (ncc), which express Snail are induced at the neural plate border and begin to migrate in response to Wnt6 and Bmp expression in the surface ectoderm and dorsal neural tube respectively.

The foundation for the anatomical and functional complexity of the vertebrate central nervous system is laid during embryogenesis. After Spemann's organizer and its derivatives have endowed the neural plate with a coarse pattern along its anteroposterior and mediolateral axes, this basis is progressively refined by the activity of secondary organizers within the neuroepithelium that function by releasing diffusible signaling factors. Dorsoventral patterning is mediated by two organizer regions that extend along the dorsal and ventral midlines of the entire neuraxis, whereas anteroposterior patterning is controlled by several discrete organizers. Organizer signals come from a surprisingly limited set of signaling factor families, indicating that the competence of target cells to respond to those signals plays an important part in neural patterning.

Patterning
The underlying principle of patterning is that cells get to know their position relative to the principal axes of the nervous system - front to back and top to bottom. In effect, each cell measures its position with respect to these orthogonal coordinates much as a map-reader figures out his or her position by measuring distance from defined points. The way this works at the molecular level is that the embryo sets up a number of localised polarizing regions in the neural tube that secrete signal molecules. In each case, the molecule diffuses away from its source to form a gradient of concentration with distance. An example of this position-sensing mechanism is the top to bottom (dorsoventral) axis of the spinal cord. The bottom part of the neural tube expresses a secreted protein Sonic hedgehog. Sonic hedgehog diffuses away from the floor plate and affects cells on the dorsoventral axis according to their distance from the floor plate. When close, Sonic hedgehog induces the expression of a gene that makes a particular type of interneuron. Further away, the now lower concentration of Sonic hedgehog induces expression of another gene making motor neurons.

Hox in vertebrates
Vertebrates, including mice, have Hox genes that are homologous to those of the fly, and these genes are clustered in discrete locations with a 3'-to5' order reflecting an anterior to posterior order of expression. There are several differences between the mouse and fly Hox genes1. there are more Hox genes on the 5' side of the mouse segment; these correspond to expression in the tail, and flies do not have anything homologous to the chordate tail. 2. in the mouse, there are four banks of Hox genes: HoxA, HoxB, HoxC, and HoxD. Vertebrates have these parallel, overlapping sets of Hox genes, which suggest that morphology could be a product of a combinatorial expression of the genes in the four Hox clusters. This means that there could be a Hox code, in which identity can be defined with more gradations by mixing up the bounds of expression of each of the genes. 3. Hox genes are crucial in the orchestration of organized growth in organisms ranging from plants to humans.

HOX IN VERTEBRATES Differentiation on the anterior posterior axis is controlled by homeotic genes. Four families of genes, called homeobox or Hox genes, control differentiation along the body axis in mice. Each family consists of 10 genes and resides on a different chromosome. Temporal and spatial expression of these genes follows the same pattern as their linear order on their chromosomes.

Figure 20.17 Hox Genes Control Body Segmentation (Part 1)

Figure 20.17 Hox Genes Control Body Segmentation (Part 2)

Patterns of HOX gene expression in the hindbrain. HOX genes are expressed in overlapping patterns ending at specific rhombomere boundaries. Genes at the 3' end of a cluster have the most anterior boundaries, and paralogous genes have identical expression domains. These genes confer positional value along the anterior-posterior axis of the hindbrain, determine the identity of the rhombomeres, and specify their derivatives.

Genes respond less rapidly; require higher [RA]s

Genes respond more rapidly at lower [RA]s

Posterior CNS

Anterior CNS

Change in Hox gene expression change in morphology along the A/P axis
The signaling mechanism for expression of these genes is a gradient of Retinoic acid (RA). The RA signal regulates the pattern of Hox expression. There is a direct correspondence between the location of the Hox gene in its cluster and its responsiveness to RA.

Hox genes affect neuronal migration and the development of the mammalian brain
E.g . Hoxa2, controls the pontine neurons' responsiveness to chemicals that attract and repel them, thus telling them where to go in the brain. The Hoxa2 gene controls expression of the receptor, Robo. Robo is bound to the chemical, Slit, which prevents migrating neurons from responding to chemoattractants. If Hoxa2 is absent , pontine neurons become insensitive to Slit signaling: the neurons ignore the repellant signal and head prematurely toward the chemoattractant, guiding them into the wrong part of the brain. Thus the pontine neurons go to the bottom of the brainstem instead of going to the cerebellum. The absence of Slit or Robo causes the same type of abnormal migrations caused by the absence of Hoxa2--further evidence that all three are integral to the same system.

Vertebrate development
Although a high degree of precision in both the spatial arrangement of neurons and their connectivity is achieved from the outset, the wiring of some parts of the nervous system is later subject to activity-dependent refinement, such as the pruning of axons and the death of neurons. These losses may appear wasteful, but it is not always possible or desirable to make a complete and perfect brain by construction alone.

References
Journals Mart E, Bovolenta P Sonic hedgehog in CNS development: one signal, multiple outputs. Trends Neurosci. 2002 Feb;25(2):89-96. Books Gilbert S.F. Developmental biology