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CHAPTER 4

Approaches to development:
experimental embryology
NORMAL DEVELOPMENT
- NORMAL
DEVELOPMENT
refers to the course of
development that the embryo
follows in standard laboratory
conditions
- There is no experimental
manipulations
- Development occurs naturally
- EMBRYONIC
DEVELOPMENT
is
predictable
- Mammals
and
birds:
Development occurs at a
standard
physiological
temperature
- Free-living
embryos
(Xenopus or zebrafish): rate
of development varies on
temperature
- Development
can
be
MATERNAL (due to the
components of the egg or
the genome of the mother)
or ZYGOTIC (due to the
components
newly
synthesized by the embryo
itself after fertilization)
TERMS TO REMEMBER
1. Anterior or cranial end
- front end of an animal
2. Posterior or caudal end
- rear end of an animal
3. Dorsal
- upper surface of an animal
4. Ventral
-lower surface of an animal
(chest area)
MICROSCOPIC SECTIONS

5. Transverse section
- Section taken across the
long axis of an animal
(separating the posterior and
anterior region)
6. Longitudinal
- parallel to the long axis
7. Sagittal
VERTICAL
longitudinal
section that is cut at the
midline
8. Parasagittal
VERTICAL
longitudinal
section that is cut at one side
of the midline
9. Frontal or coronal section
- HORIZONTAL longitudinal
section that separates the
dorsal and ventral sides
STAGE SERIES
- All models used for laboratory
work have published stage
series
- Describes the course of
development as a number of
standard stages (stages the
embryo goes through as it
develops) identified through
external features using a
dissecting microscope
THE FATE MAP
- Diagram that shows what will
become of each REGION of
the embryo in the course of
NORMAL development
1. Where will it move?
2. How will it change shape?
3. What structures will it
form?
- FATE MAP changes from
stage to stage due to
morphogenetic movements
and growth

The precision of a fate map


depends on how much
random cell mixing occurs
in development
no mixing occurs as like in the
Caenorhabditis elegans, it will
be based on CELLULAR
LEVEL
FATE MAP does not indicate
anything
about
developmental commitment
(commitment
is
usually
acquired through a series of
intercellular interactions)
ALL PARTS OF EMBRYO
have a fate throughout
development

MOSAIC
- Experimentally isolated parts
DEVELOPS according to the
fate map
REGULATIVE
- Isolate parts form MORE
STRUCTURES expected from
the fate map
- Depends on the double
gradient system
Ex: ADMP-Chordin system in
Xenopus
How are fate maps constructed?
1. Label SINGLE cells or regions
of embryos
2. Locate the position and shape
of the labeled patch at a later
stage of development
Labeling can be
a. Application of extracellular
label to a patch of cells
b. Injection of an intracellular
label to one cell

Ex: Injecting to two early


blastomeres of a Xenopus
embryo mRNA for green
fluorescent protein (GFP) then
locate the labeled domains at
NEURULA stage
c. Grafting a labeled tissue to
replace an exactly equivalent
piece in the host embryo
CLONAL ANALYSIS
- Form of fate mapping wherein
a single cell is labeled by
determination
- Position and cell types of the
progeny (of the single cell) is
identified at a later stage
Labeling can be
a. Injection of one cell with a
lineage label (used for
large cells and organisms
that
do
not
grow
significantly)
ex: Xenopus or zebrafish
or sea urchin
b. Introduction into a single
cell a genetic label that will
persist without dilution
- Can be the insertion by
replication-incompetent
retrovirus
or
genetic
recombination even that
yields a visible marker
ex: Mouse and chick
- CLONAL ANALYSIS: decide
whether a cell is committed to
form a particular structure or
cell type at the time of labeling
* a label applied early can
span up to A and B but if the
label is applied later, it will
only span either A or B
- CLONAL ANALYSIS can
prove lack of commitment but
not presence of commitment

USUALLY USED in analysis


Drosophila
segmentation
and of vertebrae hindbrain
patterning
COMPARTMENT: indicate a
region in an embryo whose
boundaries are boundaries of
clonal restriction
if
there
is
a
compartment, no cells may
enter or no cells may leave
- A compartment usually
corresponds to a visible
structure
such
as
a
segment or organ rudiment
(organ not fully developed)
and is maintained either by
physical boundaries to cell
migration such as basement
membranes or by differential
adhesion of the cells of the
compartment compared to
those outside
Cells
in
the
compartment stick to each
other

DEVELOPMENTAL COMMITMENT
- Formerly uncommitted cells
become committed cells to
form body parts of cell
types
- Commitment
is
the
combination of transcription
factors present in the cell so it
can be visualized directly by
observing the expression of
the relevant genes using in
situ hybridization
1. Specification
- a cell or tissue is said to be
specified
to
become
a
PARTICULAR STRUCTURE

It will still develop into a


particular structure even if the
cell or tissue is isolated from
the embryo
Specification map
* shows that the cells have
been programmed to do or be
at
this
particular
developmental stage
SPECIFICATION need not
be the same as the fate in
normal development
Ex: neural plate of a Xenopus
blastula may not differeniate
into a neuroepithelium but into
epidermis when cultured in
isolation
(in
order
to
form
neuroepitheliym,
inductive
signal from the mesoderm is
needed)

2. Determination
- Commitment is irreversible
- It will continue to develop
autonomously even if is
moved to any other region
of the embryo
- Pathway of development is
UNALTERED
- Cells
have
lost
their
responsiveness
or
competence to the singals
that originally turned on the
relevant combination of
transcription factors
1. Orthotopic graft
- Graft to the same
position of another embryo
2. Heterotopic graft
- Graft to the different
position in the host
- Test for determination
Potency
- range of possible cells types
or structures into which a

particular cell population may


develop
Embryonic stem cells
- cells that can form any cell
type in the body
- pluripotent and tissue specific
stem cells
- multipotent
CYTOPLASMIC DETERMINANTS
- Determinant is a substance
or substances located in part
of an egg or blastomere
- The determinant guarantees
the assumption of a particular
state of commitment by the
cells that inherit is during
cleavage (determinants for
morphogenesis)
- Cell division is asymmetrical
and two daughter cells will
follow different pathways od
development
- Determinants are important
during the earliest stages of
embryonic development: first
two or three distinctly
specified regions in embryo
are established
Ex: bicoid and nanos mRNA
in the Drosophila egg
PAR3,PAR6 and aPKC in the
C. elegans
INDUCTION
- The extracellular signals are
called inducing factors
- Growth factors, cytokines or
hormones
- Competence is the ability to
respond to an inductive signal
- Signal
transduction
pathway is also coupled to

the regulation of transcription


factors
Ex: Xenopus embryo the
mesoderm is induced from
animal hemisphere tissue in
response to the nodals
emitted from the vegetal
region
1. Instructive Induction
responding tissue has a
choice before it (mesoderm or
endoderm), and in normal
development the interaction
results in an increase in
complexity of embryo
There are two different types
of
instructive
induction
depends on
a. regional specification: if
the signaling center lies
at one end of a cell sheet
and is the source of a
concentration of a signal
substance
* Threshold response to
the
difference
concentrations

morphogen
Ex of morphogens: sonic
hedgehod protein in the
neural tube and the limb
bud, active BMP (bone
morphogenic protein in
Xenopus)
or
Dpp
(Decapentaplegic protein)
in Drosophila
b. If the signaling center lie
in one cell sheet and
responding
cells
in
another

single
threshold response
ex: induction of nasal, lens
and otic placodes
2. Appositional induction
- only one threshold
response would be made

by the responding tissue


and the inducing factor
would not be called a
morphogen
3. Permissive induction
signal is necessary for the
successful self-differentiation
of the responding tissue but
cannot
influence
the
developmental
pathway
selected
important
in
late
development
ex: development of kidney
INDUCTIVE
SIGNALS
DIFFUSE
THROUGH
EXTRACELLULAR SPACE
CYTONEMES:
transport
through FINE intercellular
processes

LATERAL INHIBITION
- Another
type
of
cell
communication
- Known in terms of the
behavior
of
Notch-Delta
system
- The individual cells from a
uniform population follow
one pathway while the
surrounding cells follow
another
Ex: neurogenesis, formation
of endocrine cells in the
epithelia of the gut
- Activator
and
inhibitor
substance
- Activator substance is short
ranged and only active
through
intercellular
contacts
- Inhibitor substance is long
ranged, moving freely with
diffusion

STOICHASTICITY IN
DEVELOPMENT
- there
is
random
determination
(random
distribution
or
random
pattern)
- DETERMINANTS
become
localized at one end of the cell
rather than the other
- Multiple cell types appear to
differentiate from a single
type of progenitor
- SYMMETRY BREAKING
THE EPIGENETIC LANDSCAPE
- Developmental commitment is
the subject of a remarkable
concept first introduced by the
embryologist and geneticist
C.H. WADDINGTON
- WADDINGTON introduced
the term EPIGENETIC as
synonym of development,
recognizing that a large
element of development
consists of the regulation of
gene expression
- The epigenetic landscape
portraying symmetry
breaking event
- Ball: cell in an embryo
- Valleys: states of
developmental commitment
- Each Bifurcation:
developmental choice
Problems
1. Diagram relates only to cell
states and does not capture
the structure of embryo
2. Exchange of inductive
signals are not represemted
3. Most developmental
choices are not symmetry
breaking events BUT
DETERMINISTIC EVENTS

controlled by inductive
signals
How is EPIGENETICS DEFINED
NOW?
- It is the understanding of gene
expression
in
terms
of
chromatin structure rather
than of DNA alone
- Importance
of
DNA
methylation as a controller of
gene
expression
and
numerous
chemical
modifications of histones and
other chromosomal proteins,
which can collectively prevent
or enable the activity of
transcription
CRITERIA FOR PROOF
1. Expression
- molecule
should
be
expressed in the right place,
right
stage
and
in
a
biologically active form
- in situ hybridization or
immunostaining
- use of retinoic acid
2. Activity
- molecule
must
have
appropriate biological activity
in a suitable test system

ex: a candidate inducing


factor should be able to evoke
the correct responses from its
target tissue
ex: the candidate cytoplasmic
determinant: it should be
possible to inject it into
another part of the cell or
blastomere and cause the
injected region to develop
along the pathway caused by
the determinant

3. Inhibition
- molecule is in vivo (inside)
then the process for white it is
thought responsible should
fail to occur
- redundancy
- inhibition may be achieved at
the DNA level by mutation of
a gene to inactivity
- Ex:
neurogenesis,
formation of endocrine cells
in the epithelia of the gut
- At RNA level, using of
antisense morpholinos or
RNAi
- At protein level, introduction of
a specific inhibitor of the
normal gene product