molecular control of brain development

7/31/2019 Molecular Control of Brain Development

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Molecular control of braindevelopment

Neuronal Patterning and

Regionalization

2012


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The Spemann Organizer

In 1924, the Ph.D. student Hilde Mangold working in the laboratoryof German embryologist Hans Spemann performed an experimentthat demonstrated that the pattern of development of cells isinfluenced 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 duringgastrulation and form the notochord (the future backbone; madeofmesoderm).

She cut out a piece of tissue from the gray crescent region of onenewt gastrula and transplanted it into the ventral side of a secondnewt gastrula.

To make it easier to follow the fate of the transplant, she used theembryo of one variety of newt as the donor and a second variety asthe recipient.
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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 two-

headed 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 toinfluence the fate of another. And because of the remarkable inductive power of the

gray crescent cells, they called this region the organizer.


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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.

Asneural tissue was derived from recipient cells, not

donor cells the transplant had altered the fate of the

overlying cells


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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.

The Organizer of Spemann and Mangold.


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Restriction of Cellular Potency.

The fate of embryonic cells is affected by both the distribution of cytoplasmic

determinants and by cleavage pattern.


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Steps during neural development

Neurogenesis

Compartmentalization

Neural differentiation Neural migration

Axonal guidance

Synaptogenesis


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Neural development in

vertebrate embryo:

Gastrulation

Blastulastage 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 theirfinal destination

Blastula stage through neurulae, highlighting gastrulation and neurulation.


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Organizing Centers:Restricted specialized areas that are crucial for the

induction of area specification

Spemanns organizer (dorsoblastopore lip)

Hensensnode (similar to Spemanns org) Roofplate and notochord become organizers

secondary organizers:

Isthmic organizer (IsO)

Anterior neural ridge (ANR)

Cortical hem


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Default model of neural induction.

Expression of signaling factors:

Bone morphogenic protein (BMP),

a TGF--like polypeptide growthfactor (PGF )expressed in ectodermon ventral side, inducing ectodermto 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.

Balance between agonists and

antagonists!

Importance of inhibition as adevelopmental regulatory

mechanism


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Signaling pathway involving BMPs

Large family of polypeptide growth factors (PGF) related to transforming growthfactor- (TGF-): BMP, activin, and GDF group members.

Heterodimer receptors, with type I & type II subunits, cytoplasmic domains withserine/theronine kinase activity.

Transforming growth factor beta (TGF-beta) and activin bind to receptor complexesthat contain two distantly related transmembrane serine/threonine kinases known asreceptor types I and II. The type II receptors determine ligand binding specificity, andeach 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 activateexpression of downstream target genes.

Inhibitory mechanisms regulate signaling:

Extracellular proteins such as chordin, tolloid, and twisted gastrulation interactwith the BMP-like ligands, regulating their diffusion through the extracellularmilieu and their ability to bind receptor

Cell surface proteins such as BAMBI inhibit signaling by binding up BMPs butfailing to transduce a signal.

Inhibitory SMADs poison the signal transduction pathway.


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Signaling transduction pathway

involving BMPs1.Activation of cytoplasmic

proteins (SMADs), whichtranslocate to nucleus toactivate expression ofdownstream target genes.2.Inhibitory mechanismsregulate signaling:

Extracellular proteins such as

chordin, tolloid, and twistedgastrulation interact with theBMP-like ligands, regulatingtheir diffusion through theextracellular milieu and theirability to bind receptorCell surface proteins such as

BAMBI inhibit signaling bybinding up BMPs but failing totransduce a signal.Inhibitory SMADs poison thesignal transduction pathway.


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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 andsegregates into the embryo,

neural crest cells emigrate

from the dorsal aspect of the

neural tube.


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Steps during neural development

Neurogenesis

Compartmentalization

Neural differentiation Neural migration

Axonal guidance

Synaptogenesis


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Early Neural Patterning:

Establishment of AP Axis

In the early stages of pattern formation, twoperpendicular 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 axialdifferences in developing structures

Position information leads to changes in geneactivity, and thus cells adopt a fateappropriate for their location


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AP polarity of vertebrate CNS

Head organizerbecomesprecordalmesoderm (PME)underneathprechordal plate

Tail organizerbecomesnotochord andsomites,underneathepichordal neuralplate

Head and tailorganizer releasefactors whichcreate a gradient.


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Neural Patterning

A/P polarity and other key organizational features are

first established by gradients ofpositional informationof a gradient of a substance or signal.

Gradient confer positional information as the relativeconcentrations correlated with distance.

In the 3-dimensional system of the embryo, the initial

establishment of A/P polarity is signalled by theorganizer (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 thenotochord.

The chordal mesoderm, which underlies the futuremidbrain, hindbrain, and spinal cord, apparentlysends out distance signals from prechordal

mesoderm.


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The candidate neural inducers, (chordin, noggin, andfollistatin) induce primitive neural tissue that appearsto be forebrain-like; chordin particularly potent.

These 3 proteins antagonize members of TGF- signallingfamily of molecules. This suggests that induction ofanterior neural plate differentiation involves inhibitorsof TGF--like signals that repress neural development.

This would be a ground state, which would be inducedto be more posterior by a 2nd signal: a transformingsignal.

In this case, a type of gradient, a ratio between activating

(noggin) and transforming signals would determinethe A/P polarity along the neuraxis.

Possible candidate posteriorizers (transforming signals)include bFGF and retinoic acid.


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Headorganizer:

BMP

Inhibitors

Cordin and

Noggin,

Wnt

inhibitors

Cerberus,

Dickkopf and

frzb1 to

"anteriorize"

neural tube

Tail organizer:

FGF, WNT, RA &BMP

inhibitors

are posteriorizing

signaling molecules


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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.


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Dorsal Ventral pattern: Notochord as organizer

Experiment using Pax gene-Left: During development, the floor plate (red) develops abovethe 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.


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Shh activity in the ventral neural

tube (blue dots) is distributed in a

ventral-high, dorsal-low profilewithin the ventral neural

epithelium.

5 classes of neurons are generated

in response to graded Shh signalling

T.M. Jessell, 2000

Sonic-hedge-hog expression by notochord & floor

plate, control of ventral patterns


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Sonic Hedge hog gene and neuron

Shh acts as a morphogen, forming a gradient in the ventral neural tube, to whichcells differentiate in a concentration- dependent fashion

SHH is a member of the hedgehog family of signalling molecules identified byhomology to the Drosophila hedgehog (HH). SHH is proteolytically cleaved toproduce two secreted proteins , a 19 kDa N-terminal protein (N-SHH) that mediatesall signalling activities in vertebrates and invertebrates and a 25 kDa C-terminalprotein (C-SHH) that possesses protease activity.

N-SHH is responsible for a number of early patterning processes; it is involved inthe control of leftright asymmetry, dorsoventral patterning of the CNS andsomites, patterning of the limb, as well as in some aspects of organogenesis

Sonic hedgehog is a secreted extracellular protein that transmits its signal bybinding to a receptor on the surface of a cell. That binding, in turn, propagates thesignal 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 thePax, Nkx, Dbx and Irx families


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(c) Cell types induced byShh 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.

(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 inthe notochord

and floor plate.

(d) Transverse

section at level

of spinal cord

(indicated inpanel c)

showing ventro-

lateral cell types

(green)

arranged with

bilateral

symmetry

around ventral

midline floorplate cells (red).


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

(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


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SHH signalling pathway

At the cell surface, SHH binds with high affinity to patched(Ptc), a 12- transmembrane protein. In mammals, twoisoforms of Ptc are encoded by Ptc1 and Ptc2, althoughPtc1 appears to be active in the CNS .

Binding of SHH to Ptc prevents the normal inhibition ofsmoothened (Smo), a seven-transmembrane protein witha topology reminiscent of G-protein-coupled receptors,which is the signalling component of the SHH-receptorcomplex.

During development of the vertebrate CNS, either inhibitionof Gi proteins or expression of a constitutively activeform of Smo is sufficient to trigger some actions of SHH.


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SHH signalling pathway

Hedgehog-interacting protein (Hip) is a type Itransmembrane protein that attenuates SHH signallingby binding N-SHH with an affinity similar to that of Ptc1

Vitronectin, an extracellular matrix glycoprotein,

enhances SHH activity during motor-neurondifferentiation, also by binding SHH directly.

Within the nucleus of the responding cell, zinc-finger

transcription factors of the Ci/GLI family (GLI13) act atthe last known step of the SHH-signal-transductionpathway , although it is still unclear whether GLI

proteins mediate all aspects of SHH signalling during

vertebrate CNS development .


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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 lastknown step of the

SHH-signal-

transduction pathway


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(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

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

(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 expressionshown on the right.

(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.


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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 interactionsbetween pairs of transcription factors

sharpen mutually exclusive expression

domains.

Right: Profiles ofhomeobox gene

expression define

progenitor zones and

control neuronal fate.


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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 andlateral 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.


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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 bythe notochord and independently for the induction of motor neurons byboth 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 intoventralized progenitors

2. a late period that extends well into S phase of the final progenitor celldivision, during which SHH drives the differentiation of ventralizedprogenitors into motor neurons.

The ambient SHH concentration during the late period determines whetherventralized progenitors differentiate into motor neurons or interneurons,

thus defining the pattern of neuronal cell types generated in the neuraltube.


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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 fromthe floor plate.

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 bycombinations of homeobox

transcription factors.

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.

d| The role of RA ingenerating a subset of

motor neurons in the

spinal cord. Retinaldehydedehydrogenase 2 (Raldh2) is expressed

in motor neurons at limb levels (red

circles). A subset of motor neurons

known as lateral motor columnneurons (LMCs) originates close to the

midline of the cord (green circles) and

then migrates through the Raldh2-

expressing 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.


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(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 neuronsdifferentiate (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).

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 andthese are the presumptive

motor neurons (mn). (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).


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In the stem zone, FGFs from theunderlying 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 dehydrogenase2 , 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 neuraltube (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 diagram of the posterior end of the

embryo where DV patterning is taking place.


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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


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Regionalization of the Nervous System

I. Segmentation

II. Developmental control genes (e.g., Hox), whichencode positional values along A/P axis.A positionalsignaling mechanism activates these genes .

E.G. In birds, At Hensons Node (similar to blastopore ofhigher animals), a strong candidate for this signal isa gradient ofretinoic acid, which regulates thepattern ofHoxgene expression.

Different Hoxgenes at specific locations respond moreor less readily to lower or higher [RA]s, through afamily of receptors, which, bound by RA, becometranscription factors.


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I. Segmentation: Subdivision of the main body axis bysegmentation provides compartments, which allocateprecursor cells into a repeated set of similarmolecules, so that developmental fields can remainsmall, and specialization of cell types and patternscan be generated as local variations on the repetitivetheme.

e.g. Mesoderm = segmented into somites, yielding

muscle groups.The neuraxis is also segmented

In the CNS, segmentation is a mechanism for specifyingpattern during development.

The earliest neurons and neural pathways are laid out instripes, which match a morphological repeat pattern

( a 2-segment repeat pattern, which has similarpatterns of development in even- or odd-numberedsegments).


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Cells are segregated by:

a. Mechanical boundaries (certain extracellular

matrix pattern, such as chondroitin SO4appear 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


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Compartmental organization of hindbrain into

rhombomeres in Zebra fish

Genes are expressed in

alternate stripes that

correspond withpresumptive

Rhombomeres.

Restriction of

movement of mitoticPrecursor cells across

interfaces.

The interfaces

betweenRhombomeres acquire

molecular and

Morphological

specialization marked

by distinct boundaries.Julie E. Cooke, Cecilia B. Moens, 2002: Schematic dorsal views of part of thedeveloping 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, 50m

(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 post-

fertilization),

(b) Gene expression domain boundaries

progressively sharpen to form straight interfaces.

At 18 somites (18 hours post-fertilization),

domains ofkrox20 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-red-

phalloidin 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 withrespect 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.


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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 bi-

directional repulsive signaling

mediated the Eph/ephringene 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.


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Stages in the compartmental organization of rhombomeres

Genes such as Krox20 and EphA4

(blue) and ephrin-B2 (pink) are

expressed in alternate, fuzzy-

edged stripes (left). Subsequently,

restriction to the movement of

mitotic precursor cells occurs at

the interfaces between newlyformed rhombomeres, which are

now sharply defined, and marked

by increased intercellular spaces.

(right)

Sharpening of boundaries and cell

lineage restriction occur through

the interaction ofEph and ephrin

molecules.


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Neurons and synapseOnce 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

longdistances. 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 helpthem 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


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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 embryotarget 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.


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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, thetransition (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.


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Axon growth, requires (A) the supply of building blocks as well as (B) cycling filaments in thegrowth 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


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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.


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S G ti


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Synapse Generatione.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.


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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 isable to receive.

Relatively little is known about the molecular mechanisms that control

dendrite development.

1. Regulation of dendritic field size andcomplexity by transcription factors:

In some cases, the "dendritic fate" of a particular neuron might be specifiedby 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 combinedaction of multiple transcription factors.


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Genetic Control of Dendrite Development in

Drosophila

Drosophila da (dendritic arborization) neurons fall into four distinctmorphological 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 morecomplex dendritic branching patterns and larger dendritic fields (classes III

and IV) express higher levels of Cut. Cut levels are a critical determinant ofda neuron class-specific dendritic morphologies.

In contrast to Cut, Spineless (Ss), the homolog of the mammalian dioxinreceptor, is expressed at similar levels in all da neurons. Studies of theepistatic relationship between Cut and Ss indicate that these transcriptionfactors 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 oftranscriptional regulators likely regulate type-specific dendritearborization patterns.

G ti C t l f D d it


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Genetic Control of Dendrite

Development in Drosophila

2. The molecular mechanism for dendritic self-avoidance and tiling. The dendritesof each da neuron show self-avoidance and tend to spread out without crossingover. Class III and class IV da neurons show tiling; i.e., there is little overlapbetween the dendritic fields of adjacent neurons of the same class because theirdendrites 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 daneuron bundle together or cross over. For the dendritic fields of different neuronsto coexist in the same space, they need to express different Dscam isoforms.

In contrast, tiling requires some cell surface recognition molecules other thanDscam to mediate the homotypic repulsion. The evolutionarily conserved proteinkinase Tricornered (Trc) and the putative adapter protein Furry (Fry) have beenidentified as important components of the intracellular signaling cascade involved

in tiling. 3. Dendrite-specific developmental regulators are a group ofdar(dendritic

arborization reduction) genes. Mutations of any of the dargenes lead to defectivedendritic arbors but normal axonal projections. There may be a total of about 20dargenes in Drosophila.

G ti C t l f D d it


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Genetic Control of Dendrite

Development in Drosophila4. 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 themaintenance of class IV da dendrites. Loss-of-function mutants of any of thosegenes cause a progressive defect in the maintenance of dendritic tiling, resulting inlarge 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, thoseneurons prune all their dendrites. Later each neuron grows a completely newdendrite for adult function. While the dendrites are being remodeled, the axonsstay largely intact.

Extension of Drosophila Work to Dendrite Development of MammalianCentral Neurons:The great majority of the genes found to affectDrosophila dendrite development have a mammalian homolog(s). Inseveral cases those homologs (for example, Dasm1, Dar3/Sar1) havesimilar function in regulating dendrite development in the mammaliancentral nervous system.

l l l


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II. Developmental Control Genes.

These genes, which encode txn factors, or signaling molecules, areexpressed in a spatially variable manner.

The Hoxgenes (homeobox family) have a clustered chromosomal

organization. The relative position of the gene reflects theexpression along the A/P axis. This expression of the Hoxgeneconfers positional value and regional identity.

The Hox genes are a set of transcription factor genes that exhibit anunusual property: These are genes that specify segment identitywhether a segment of the embryo will form part of the head,thorax, or abdomen, for instanceand they are all clusteredtogether in one (usually) tidy spot. Within that cluster, there is evenfurther evidence of order.

Unlike most genes, however, the order of Hox genes in the genomeactually holds meaning. Hox code represents is a somewhat digital

mechanism for regulating axial patterning. By mixing and matchingcombinations of the expression of a small number of Hox genes,organisms generate a greater range of morphological possibilities
http://www.nature.com/scitable/topicpage/Genetic-Signaling-Transcription-Factor-Cascades-and-Segmentation-1058http://www.nature.com/scitable/topicpage/Genetic-Signaling-Transcription-Factor-Cascades-and-Segmentation-1058http://www.nature.com/scitable/topicpage/Genetic-Signaling-Transcription-Factor-Cascades-and-Segmentation-1058


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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 ofexpression in the fly body. The gene found on the left or 3' end ofthe DNA strand, denoted lab (labial), is expressed in the head; onthe 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 homeotictransformationsin other words, one body part develops intoanother. A famous example is the Antennapedia mutant, in whichlegs develop on the fly's head instead of antennae.

The Hox genes are early actors in the cascade of interactions thatenable the development of morphologically distinct regions in asegmented animal.e.g. the activation of a Hox gene from the 3' endis one of the earliest triggers that lead the segment to develop intopart of the head.

Genomic Organization of the Hox Gene Cluster


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g

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-Hoxhomeodomain 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.


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5 3Genes respondmore rapidly at

lower [RA]s

Genes respondless rapidly; require

higher [RA]s

Posterior CNS Anterior CNS

Change in Hoxgene expression

change in morphology along the A/P axisThe signaling mechanism for expression of these genes is a gradient of

Retinoic acid (RA). The RA signal regulates the pattern ofHoxexpression.

There is a direct correspondence between the location of the Hoxgene in its

cluster and its responsiveness to RA.


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Patterning of the brain and spinal cord through


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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 ofRA and FGF signaling.


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Regional specification in the developing brain

Three-vesicle state Five-vesicle state

Rh b th l t bdi i i titi th hi db i ith li


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

CHICK.


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Hox gene expression domains in the CNS


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Hox gene expression domains in the CNS

Nested domains of homeotic genes along the AP axis of the Drosophila and mouse CNS.

Hoxgenes 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


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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 themesoderm 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).

The organizer does NOT induce the centralSUMMARY


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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 brainand 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

SUMMARY


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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 firston Wnt transcription factors. Theirmessenger RNAs were deposited by themother in the vegetal pole of the egg. After

fertilization and formation of the graycrescent, they migrated into the graycrescent region (destined to become theorganizer) where they were translated intoWnt protein.

Wnt protein accumulation on the dorsal sideof the embryo unleashes the activity ofNodal a member of the TGF- family.Nodal induces these dorsal cells to begin

expressing the proteins of Spemann'sorganizer.

ORGANIZERS IN DROSOPHILA Proteins similar in structure to the bone

morphogenetic proteins and also to chordinare found in Drosophila.

The role ofBMP-4 is taken by a relatedprotein encoded by the decapentaplegicgene (dpp).

The role ofchordin is taken by a related

protein called SOGencoded by the genecalled short gastrulation.

In Drosophila, DPP is produced in the dorsalregion of the embryo and SOG is producedin the ventral region.
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Difference in Drosophila and Humans

The actions of SOG on overlying cells are the same asin Xenopus; that is, the SOG protein prevents the DPPprotein from blocking the formation of the centralnervous system. The result in Drosophila is that its

central nervous system forms on the ventral side of theembryo, not on the dorsal! One of the distinguishingtraits of all arthropods (insects, crustaceans, arachnids)as well as many other invertebrates, such as theannelid worms, is a ventral nerve cord.

Chordates, including all vertebrates, have a dorsal(spinal) nerve cord.
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Human nervous system develpment

Cell differentiation begins with the emergence of the cells in thethree 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 centralnervous system begin as a further differentiation of ectoderm cellsinto a layer known as the neural plate.

Neural plate formation is induced by chemical signals from themesoderm (evidently peptides with molecular weight less than1,000). The neural plate folds and differentiates into neural crestcells and a neural tube. The neural crest cells become theperipheral 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


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Neurulation

The neural plate forms after

gastrulation is completed.

The neural tube narrows

along its medial-lateral

Axis. The plate begins torole into a tube. The

cells at the midline produce

a medial hinge point

(MHP).

As the tube forms andsegregates into the embryo,

neural crest cells emigrate

from the dorsal aspect of the

neural tube.


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Neurulation Genes give dorsalventral position information.

Sonic hedgehogis an example of a dorsalventralgene that is expressed in the notochord andinduces cells in the overlying neural tube tobecome ventral spinal cord cells.

Another family of homeobox genes, the Pax genes,are important in nervous system and somitedevelopment. Pax3 is expressed in neural tubecells that will become dorsal spinal cord cells.

Pax3 and sonic hedgehog interact to determinedorsalventral differentiation of the spinal cord.

Neurulation.


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(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 neuralplate border and begin to

migrate in response to Wnt6

and Bmp expression in the

surface ectoderm and dorsal

neural tube respectively.


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Th f d i f h i l d


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The foundation for the anatomical and

functional complexity of the vertebrate

central nervous system is laid during

embryogenesis.

After Spemann's organizer and itsderivatives have endowed the neural plate

with a coarse pattern along its

anteroposterior and mediolateral axes, this

basis is progressively refined by the

activity ofsecondary 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.


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Patterning

The underlying principle of patterning is that cells get to know their position relative tothe 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.

b


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Hox in vertebrates

Vertebrates, including mice, have Hox genes that are homologous to thoseof the fly, and these genes are clustered in discrete locations with a 3'-to-5' order reflecting an anterior to posterior order of expression.

There are several differences between the mouse and fly Hox genes-

1. there are more Hox genes on the 5' side of the mouse segment; thesecorrespond 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 Hoxgenes, which suggest that morphology could be a product of acombinatorial expression of the genes in the four Hox clusters. Thismeans 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 ofthe genes.

3. Hox genes are crucial in the orchestration of organized growth inorganisms ranging from plants to humans.


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Differentiation on the anterior

posterior axis is controlled byhomeotic genes.

Four families of genes, called

homeobox or Hox genes, control

differentiation along the body

axis in mice.

Each family consists of 10 genesand resides on a different

chromosome.

Temporal and spatial expression

of these genes follows the same

pattern as their linear order on

their chromosomes.

HOX IN VERTEBRATES

Figure 20.17 Hox Genes Control Body Segmentation (Part 1)


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Figure 20.17 Hox Genes Control Body Segmentation (Part 2)


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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.


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5 3Genes respondmore rapidly atlower [RA]s

Genes respondless rapidly; require

higher [RA]s

Posterior CNS Anterior CNS

Change in Hoxgene expression

change in morphology along the A/P axisThe signaling mechanism for expression of these genes is a gradient of

Retinoic acid (RA).

The RA signal regulates the pattern ofHoxexpression.

There is a direct correspondence between the location of the Hoxgene in its

cluster and its responsiveness to RA.

Hox genes affect neuronal migration and the


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Hox genes affect neuronal migration and the

development of the mammalian brain

E.g . Hoxa2, controls the pontine neurons' responsiveness tochemicals that attract and repel them, thus telling them where togo in the brain.

The Hoxa2 gene controls expression of the receptor, Robo. Robo isbound to the chemical, Slit, which prevents migrating neurons fromresponding to chemoattractants.

If Hoxa2 is absent , pontine neurons become insensitive to Slitsignaling: the neurons ignore the repellant signal and headprematurely toward the chemoattractant, guiding them into thewrong part of the brain. Thus the pontine neurons go to the bottomof the brainstem instead of going to the cerebellum. The absence ofSlit or Robo causes the same type of abnormal migrations causedby the absence of Hoxa2--further evidence that all three areintegral to the same system.


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V t b t d l t


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Vertebrate development

Although a high degree of precision in both thespatial arrangement of neurons and theirconnectivity is achieved from the outset, thewiring of some parts of the nervous system islater subject to activity-dependentrefinement, such as the pruning of axons andthe death of neurons. These losses may

appear wasteful, but it is not always possibleor desirable to make a complete and perfectbrain by construction alone.


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R f


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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
http://www.ncbi.nlm.nih.gov/pubmed?term=Mart%C3%AD%20E[Author]&cauthor=true&cauthor_uid=11814561http://www.ncbi.nlm.nih.gov/pubmed?term=Mart%C3%AD%20E[Author]&cauthor=true&cauthor_uid=11814561http://www.ncbi.nlm.nih.gov/pubmed?term=Bovolenta%20P[Author]&cauthor=true&cauthor_uid=11814561http://www.ncbi.nlm.nih.gov/pubmed/11814561http://www.ncbi.nlm.nih.gov/pubmed/11814561http://www.ncbi.nlm.nih.gov/pubmed/11814561http://www.ncbi.nlm.nih.gov/pubmed/11814561http://www.ncbi.nlm.nih.gov/pubmed/11814561http://www.ncbi.nlm.nih.gov/pubmed/11814561http://www.ncbi.nlm.nih.gov/pubmed?term=Bovolenta%20P[Author]&cauthor=true&cauthor_uid=11814561http://www.ncbi.nlm.nih.gov/pubmed?term=Bovolenta%20P[Author]&cauthor=true&cauthor_uid=11814561http://www.ncbi.nlm.nih.gov/pubmed?term=Bovolenta%20P[Author]&cauthor=true&cauthor_uid=11814561http://www.ncbi.nlm.nih.gov/pubmed?term=Mart%C3%AD%20E[Author]&cauthor=true&cauthor_uid=11814561http://www.ncbi.nlm.nih.gov/pubmed?term=Mart%C3%AD%20E[Author]&cauthor=true&cauthor_uid=11814561http://www.ncbi.nlm.nih.gov/pubmed?term=Mart%C3%AD%20E[Author]&cauthor=true&cauthor_uid=11814561http://www.ncbi.nlm.nih.gov/pubmed?term=Mart%C3%AD%20E[Author]&cauthor=true&cauthor_uid=11814561http://www.ncbi.nlm.nih.gov/pubmed?term=Mart%C3%AD%20E[Author]&cauthor=true&cauthor_uid=11814561

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