By:

Dr. Khaled El Masry

Assistant Lecturer of Human Anatomy & Embryology

Mansoura Faculty of Medicine, Mansoura University,

Mansoura , Egypt.

Molecular Embryology

20134/28/2013

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All Data and Diagrams included

in this presentation are basically

derived from our highly

valuable reference of

Embryology

(Langman's Medical

Embryology 12th ed. - T.

Sadler (Lippincott, 2012)

, recommending you all to refer

to it for more details….

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Introduction

Molecular Biology has opened the doors to new ways

to study Embryology and to enhance our

understanding of Normal and Abnormal development.

Sequencing the Human Genome, together with

creating techniques to investigate gene regulation at

many levels of complexity, has taken Embryology to

the next level.

Thus, from Anatomical to Biochemical to

MOLECULAR level, the story of Embryology has

progressed, and each chapter has enhanced our

knowledge.

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Genes are contained in a complex of DNA and proteins called chromatin, and

its basic unit of structure is the nucleosome. Chromatin appears tightly coiled

as beads of nucleosomes on a string and is called heterochromatin.

For transcription to occur, DNA must be uncoiled from the beads as

euchromatin. Genes reside within strands of DNA and contain regions that can

be translated into proteins, called exons, and untranslatable regions, called

introns.

A typical gene also contains :

1. a promoter region that binds RNA polymerase for the initiation of transcription;

2. a transcription initiation site, to designate the first amino acid in the protein;

3. a translation termination codon; and

4. a 3′ untranslated region that includes a sequence (the poly A addition site)

that assists with stabilization of the mRNA.

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The RNA polymerase binds to the promoter region that usually

contains the sequence TATA, the TATA box. Binding requires

additional proteins called transcription factors. Methylation of

cytosine bases in the promoter region silences genes and

prevents transcription.

Different proteins can be produced from a single gene by the

process of alternative splicing that removes different introns

using spliceosomes. Proteins derived in this manner are called

splicing isoforms or splice variants. Also, proteins may be

altered by post- translational modifi cations, such as

phosphorylation or cleavage.

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Induction is the process whereby one group of cells or tissues (the

inducer) causes another group (the responder) to change their fate.

The capacity to respond is called competence and must be conferred

by a competence factor.

Many inductive phenomena involve epithelial– mesenchymal

interactions.

Signal transduction pathways include a signaling molecule (the

ligand) and a receptor.The receptor usually spans the cell membrane

and is activated by binding with its specific ligand.

Activation usually involves the capacity to phosphorylate other

proteins, most often as a kinase. This activation establishes a cascade

of enzyme activity among proteins that ultimately activates a

transcription factor for initiation of gene expression.

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Cell-to-cell signaling may be paracrine, involving diffusable

factors, or juxtacrine, involving a variety of nondiffusable factors.

Proteins responsible for paracrine signaling are called paracrine

factors or growth and differentiation factors (GDFs).

There are four major families of GDFs:

FGFs, WNTs, hedgehogs, and TGF-bs.

In addition to proteins, neurotransmitters, such as serotonin (5HT)

and norepinephrine, also act through paracrine signaling, serving as

ligands and binding to receptors to produce specific cellular

responses.

Juxtacrine factors may include products of the extracellular

matrix, ligands bound to a cell’s surface, and direct cell-to-cell

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Molecular Regulation ofGastrulation

(Establishment of the Body Axes)

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Establishment of the body axes;

anteroposterior, dorsoventral, and left-right, takes

place before and during the period of gastrulation.

Cells at the prospective cranial end of

the embryo in the anterior visceral

endoderm (AVE) express the

transcription factors OTX2, LIM1, and

HESX1 and the secreted factor

cerberus that contribute to head

development and establish the

cephalic region.Goosecoid, expressed in the node, regulates chordin

expression, and this gene product, together with noggin and

follistatin, antagonizes the activity of BMP4, dorsalizing mesoderm

into notochord and paraxial mesoderm for the head region.Once the streak is formed and gastrulation is progressing, BMP4 is

secreted throughout the bilaminar disc and acts with FGF to

ventralize mesoderm into intermediate and lateral plate mesoderm.

Later, expression of the Brachyury (T) gene antagonizes BMP4 to

dorsalize mesoderm into notochord and paraxial mesoderm in caudal

regions of the embryo.

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FGF8, secreted by the node and

primitive streak, establishes

expression of Nodal, a member of

the TGF-b superfamily, and the nodal

protein then accumulates on the left

side near the node.

Later, as the neural plate starts to

form, FGF8 induces expression of

Nodal and LEFTY-2 in the lateral

plate mesoderm, whereas LEFTY-1

is expressed on the left side of the

ventral aspect of the neural tube.

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These signals are dependent upon

serotonin (5HT). Products from the

Brachyury (T) gene, expressed in the

notochord, also participate in induction of

these three genes.

In turn, expression of Nodal and

LEFTY-2 regulates expression of the

transcription factor PITX

2, which, through further downstream

effectors, establishes left-sidedness.

SHH, expressed in the notochord, may

serve as a midline barrier and also

represses expression of left-sided genes

on the right. Expression of the

transcription factor Snail may regulate

downstream genes important for

establishing right-sidedness.

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Cells migrating more posteriorly

through the node and most cranial

aspect of the streak will form

paraxial mesoderm (pm;

somitomeres and somites);

Fate map established during

gastrulation

Cells migrating at the most

cranial part of the node will

form the notochord (n);

Cells migrating through the next portion

of the streak will form intermediate

mesoderm (im; urogenital system);

Cells migrating through the more caudal

part of the streak will form lateral plate

mesoderm (lpm; body wall);

Cells migrating through the most

caudal part will contribute to

extraembryonic mesoderm (eem;

chorion).

Molecular Regulation ofGenital DuctDevelopment

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SOX

9

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Estrogr

n

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Molecular Regulation ofPharyngeal Arches

Development

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Crest cells originating from Rhombomeres migrate to specific

arches

Patterning of pharyngeal

arches

This is regulated

by

1st Arch 2nd to 6th Arch

By

OTX 2 gene

Midbrainand ForebrainExpressed in

regions and migrate with crest cells to

1st arch.

By

HOX genes

in HindbrainExpressed in

specific overlapping

pattern

There may be

an interaction

with HOX

genes in 1st

arch

patterning

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HOX

genes

Crest cells alone can’t maintain HOX genes

expression but require interaction with

mesoderm of the pharyngeal arches

HOX expression is

regulated by

SHH

Upstream regulator

expressed in the

arches

Regulate HOX expression in concentration

RAREdependent manner via

Retinoi

c Acid

: ( Retinoic Acid Response Elements)RARE

Binding sites for retinoic acid in promoter regions of HOX genes4/28/2013

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Patterns of HOX gene expression in the

hindbrain. HOX genes are expressed in overlapping

patterns

ending at specifi c 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

anteriorposterior

axis of the hindbrain, determine the identity of

the rhombomeres, and specify their derivatives.

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Molecular Regulation ofSpinal Cord& Brain

Development

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Forebrain

&

Midbrain

Inhibition of BMP 4

Chordin

Noggin

Follistati

n

B

y

Expression of ( OTX 1,2 , EMX1, EMX2) genes in specific

& overlapping pattern

Differentiation of Forebrain & Midbrain

Regions

Once Boundaries of Forebrain & Midbrain Regions are

established

2 additional Organizing Centers appear

ANR

( at the junction of cranial border of

neural plate & non- neural

ectoderm

Isthmus

( at the junction Midbrain &

Hindbrain)

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AN

RFGF

8

BF

1

Regulate

Development of

Telencephalon

Induce expression of

Ishmu

s

FGF

8Induce expression of

WNT 1

EN

1

EN

2

Regulate

Development of

Tectum &

Cerebellum

Regulate

Development of

Cerebellum only

Interact with

EN1 & EN2

to regulate

development

of the region

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Isthmus

FGF

8

EN

1

EN

2

WNT 1

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HindbrainWNT 3a & FGF

Induce differentiation of

Hindbrain region into 8segments called

Rhombomeres

Express HOX

genes

Expressed in overlapping pattern

Determine identity of these Rhombomeres & specify their

derivatives

Retinoic Acid Organizes Craniocaudal Axis

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Patterns of HOX gene expression in the

Hindbrain:

HOX genes are expressed in overlapping patterns

ending at specifi c 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

anteriorposterior

axis of the hindbrain, determine the identity of

the rhombomeres, and specify their derivatives.

Spinal

Cord

Induced byWNT 3a &

FGF

Neural plate in spinal cord region

expresses the following

Transcription Factors

(PAX3, PAX7, MSX1, MS

X2)

This expression is

controlled by

SHH (secreted by prochordal

plate)

Inhibit their

expression

Ventralize the Neural plate

Motor Neurons formation

BMP4,7(secreted by Non-neural

Ectoderm)

Upregulate PAX3,7

Dorsalize the Neural plate

Sensory Neurons

formation

needed for

Neural

Crest cells

formation

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Molecular Regulation ofLIMB

Development

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Limb outgrowth is initiated by FGF10 secreted by

lateral plate mesoderm in the limb-forming regions

Once outgrowth is initiated, the AER is induced by

BMPs and restricted in its location by the gene

Radical fringe expressed in dorsal ectoderm. In

turn, this expression induces that of SER2 in cells

destined to form the AER.

After the ridge is established, it

expresses FGF4 and FGF8 to maintain

the progress zone, the rapidly

proliferating mesenchyme cells adjacent

to the ridge.

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Anteroposterior patterning of the limb is controlled

by cells in the ZPA at the posterior border.

These cells produce retinoic acid (vitamin A), which

initiates expression of SHH, regulating patterning.

The dorsoventral limb axis is directed by

WNT7a, which is expressed in the dorsal

ectoderm.

This gene induces expression of the

transcription factor LMX1 in the dorsal

mesenchyme, specifying these cells as

dorsal.

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Longitudinal section through the

limb bud of a chick

embryo, showing a core of

mesenchyme cover by a layer of

ectoderm that thickens at the distal

border of the limb to form the AER.

In humans, this occurs during the

fifth week of development.

External view of a chick limb at

high magnification showing the

ectoderm and the specialized

region at the tip of the limb called

the AER

Bone type and shape are regulated by HOX genes, whose

expression is determined by the combinatorial expression of

SHH, FGFs, and WNT7a.

HOXA and HOXD clusters are the primary determinants of bone

morphology.

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Molecular Regulation ofEYE

Development

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The eyes begin to develop as a pair of outpocketings that will become the optic vesicles on each side of the forebrain at the end of the fourth week of development .

The optic vesicles contact the surface ectoderm and induce lensformation.

When the optic vesicle begins to invaginate to form the pigment and neural layers of the retina, the lens placode invaginates to form the lens vesicle.

Through a groove at the inferior aspect of the optic vesicle, the choroid fissure, the hyaloid artery (later the central artery of the retina) enters the eye .

Nerve fibers of the eye also occupy this groove to reach the optic areas of the brain.

The cornea is formed by:

(a) a layer of surface ectoderm,

(b) the stroma, which is continuous with the sclera, and

(c) an epithelial layer bordering the anterior chamber

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The master gene for

Eye DevelopmentPAX

6

Expressed in the single eye field at the neural

plate stage.

SHH

Separate the

eye field into

two optic

primordia.

upregulates

PAX2 expression in

the optic stalks

while

downregulating

PAX6.

Restricting

(PAX6)expressio

n to the optic cup

and lens

Epithelial–mesenchymal interactions between prospective lens

ectoderm, optic vesicle, and surrounding mesenchyme then

regulate lens and optic cup differentiation.

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Molecular Regulation ofTOOTH

Development

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Teeth develop from epithelial–mesenchymal interactionsbetween oral epithelium and neural crest–derivedmesenchyme.

Enamel is made by ameloblasts.

It lies on a thick layer of dentin produced byodontoblasts,

a neural crest derivative.

Cementum is formed by cementoblasts, anothermesenchymal derivative found in the root of the tooth.

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Enamel Knot(Circumscribed

Region of dental

epith. at tips of tooth

buds

The organizer for the

TOOTH

Development is

Express

FGF 4 BMP 4

Regulate

outgrowth of the

cap.

Regulate timing of

apoptosis in Knot

cells 4/28/2013

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http://www.imeg.kumamoto-u.ac.jp/en/index.html

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