Download Gene Regulation and Development

Gene Regulation and
Development
Making of
the Complex Living things
• In the development of multicell organisms, a single-cell
zygote gives rise to many different cell types.
– Each type has different structure, under taking different chemical
and biologic reactions and performs corresponding functions.
– All these events are controlled by sets of genes in the cell and
influenced by surrounding stimuli.
• Body system of multicell organisms
–
–
–
–
Cells of similar types are organized into tissues
Tissues with associated function into organs
Organs of associated function into organ systems
Organ systems coordinately to support the whole organism
• Process of embryonic development must give rise not only
to the cells of different types but also to higher-level
structures and coordinated functions.
How life begin…
• An organism arises from a fertilized egg as the
result of three interrelated processes: cell
division, cell differentiation, and cell
morphogenesis.
• During development, cells becomes polarized,
starts divide unevenly, reprograms gene
expression and become specialized in structure
and function, a process called differentiation.
• The processes of morphogenesis is forming of
shapes, give cells, tissues and organs their
proper shape to fit their corresponding functions.
• All these processes are part of development.
Embryonic Development
• Fertilization results in a zygote and triggers
embryonic development
Middle
piece
Neck
Head
Plasma membrane
Tail
Mitochondrion
(spiral shape)
Nucleus
Acrosome
1 The sperm
approaches
the egg
2 The sperm’s
acrosomal enzymes
digest the
egg’s jelly
3 Proteins on the
coat
sperm head bind
to egg receptors
SPERM
4 The plasma membranes
of sperm and egg fuse
Sperm
head
•
•
Only one sperm will
penetrate this human egg
to initiate the fertilization
There is a selection of
sperm quality
5 The sperm
nucleus
enters
the egg
cytoplasm
Nucleus
Acrosome
Acrosomal
Plasma
membrane enzymes
6
A fertilization
envelope forms
Receptor protein
molecules
Plasma
membrane
Jelly
coat
(Zona
Pellucida)
Vitelline
layer
Cytoplasm
EGG CELL
Sperm
nucleus
Egg
nucleus
7 The nuclei
of sperm
and egg fuse
Zygote
nucleus
In mammals, fertilization occurs in the oviduct. Sperm
fuses with oocyte in metafase 2, completes meiosis
and then two parenteral pronuclei fuse to form the
diploid zygote.
Cleavage starts
Fertilization
of ovum
Ovary
Oviduct
Secondary
oocyte
Ovulation
Blastocyst
(implanted)
Endometrium
Uterus
Cell Division
•
Cleavage is the first major step of
embryonic development
– It is the rapid succession of cell
divisions
– It creates a multicellular embryo
– It partitions the multicellular
embryo into developmental
regions
– Cells and embryo become
polarized
•
Cleavage is Controlled by cyclins
and cyclin-dependent kinases
(Cdks)
– During cleavage, the zygote is
divided into smaller & smaller cells
called blastomeres
– Embryo volume does not change
Blastomeres
• Non-differentiated cells and can give rise
to any tissue.
– Stem cells continue to divide and remain
undifferentiated.
– Totipotent: cells that can give rise to any
cell types
– Pluripotent: cells that can give rise to
multiple cell types
– Tissue-specific: can give rise to only one
or fewer cell types
Blastocyst
ENDOMETRIUM
•
Cell divide and cell number
increase continueously for 5-6
days (3-4 days in mice) to
produce
– the first morphologically
differentiated structure
– The first two different cell types
with totally different gene
expression patterns
•
Inner cell mass
Trophoblast
The blastocyst consists of:
– Outer layer(Trophoblast
cell )_which secretes enzymes
to enable the blastocyst to
implant on endometrium
surface and later forms
placenta
– Inner layer (Inner Cell Mass,
which is undifferentiated and a
general source of embryonic
stem cell)_which develop to
complete body parts.
Cavity
Antibody
against Oct4
Trophoblast
ES Cell Culture
Once sperm and egg cell fuse (zygote), cell start to divide and differentiate.
The first differentiated structure: a blastocyst.
The inner cell mass of the blastocyst develops into embryo.
Embryonic stem-cell
culture
Inner cell
mass
Egg
Sperm
Blastocyst
Embryo
Embryonic stem cells (ES cells) are
isolated from the inner cell mass
Determination
• The earliest changes that set a cell to a
specialized stage without turning back at
molecular level.
– Can only be “seen” by experiment
– Cells occupy a specific location in the embryo
– They are not “naturally” totipotent any more
• Molecular changes that drives the process in the
embryo is termed determination, the beginning
of differentiation.
• During embryonic development, cells become
obviously different in structure and function as
they differentiate.
Cell Differentiation
• Gene expression is switched to a new program
– Cells initiate developmental changes by using new
transcriptional factors and give a totally different
pattern of gene expression
• Cells become committed to a particular
developmental pathway:
– via genetic program under the differential inheritance
of cytoplasmic determinants
– via environmental cell-cell interactions
• Cells are “morphologically” distinguishable by
different size and shape
Differentiated Cells
• These cells produce the proteins that allow them to carry
out their specialized roles in the organism.
– For example, only lens cells devote 80% of their capacity
for protein synthesis to making just one type of proteins,
crystallins.
• These form transparent fibers that allow the lens to transmit and focus
light.
– Similarly, skeletal muscles cells have high concentrations
of proteins specific to muscle tissues, such as musclespecific version of the contractile protein myosin and the
structural protein actin.
• They also have membrane receptor proteins that detect signals from
nerve cells.
Morphogenesis
Polar Cell division
The orientation of the mitotic spindle determines the plane of cell division in
eukaryotic cells
-If spindle is centrally located, two equal-sized daughter cells will result
-If spindle is off to one side, two unequal daughter cells will result
Cell shape and size
In animals, cell differentiation is accomplished by profound changes in cell size and
shape
-Nerve cells develop long processes called axons
-Skeletal muscles cells are large and multinucleated
Cell death
-Necrosis is accidental cell death in group, not cell-specific
-Apoptosis is programmed cell death, can happen in individual cells
-Is required for normal development in all animals
-“Death program” pathway consists of:
-Activator, inhibitor and apoptotic protease
Cell migration
-Cell movement involves adhesion and loss of adhesion between cells and substrate
-Cell-to-cell interactions are often mediated through cadherins
-Cell-to-substrate interactions often involve complexes between integrins and the
extracellular matrix (ECM)
Gastrulation produces a three-layered
embryo
• The second major step of embryonic
development, produces a three-layered embryo
–
–
–
–
–
It happens after implantation
The size of embryos starts to grow
It adds more cells and volume to the embryo
It sorts all cells into three distinct cell layers
The embryo is transformed from the blastula into the
gastrula
• The three layers
– Ectoderm, the outer layer
– Endoderm, an inner layer, give rise to the embryonic
digestive tract
– Mesoderm, which partly fills the space between the
ectoderm and endoderm
Embryonic tissue layers begin to develop into specific tissues
and organ systems after gastrulation
The embryo floats in amniotic cavity, chorion and
embryonic mesoderm forms the placenta
Functions of the chorion:
1. Provide nutrients and O2 to
the fetus
2. Secrete hormones into the
mother to help retain the
fetus
3. Repress the mother’s
immune response to prevent
rejection of the fetus
4. The placenta’s chorionic villi
absorb food and oxygen
from the mother’s blood
Placenta
Allantois
Mother’s blood
vessels
Amniotic
cavity
Yolk sac
Embryo
Chorion
Chorionic
villi
Embryonic induction initiates
organ formation
• Induction is the mechanism by which one group of cells
influences the development of tissues and organs from
ectoderm, endoderm, and mesoderm
– Adjacent cells and cell layers use chemical signals to influence
differentiation
– Chemical signals turn on a set of genes in the receiving cells and
leads to differentiation into a specific tissues from these cells
• Pattern formation
– Pattern formation is the emergence of structures in their correct
relative positions
– It often involves the response of cells and their genes to spatial
gradient of chemicals
Ectoderm
•
Induction:
Tissues and organs take shape in a
developing embryo as a result of
–
–
–
–
Cell division
Cell shape changes
Cell migration
Programmed cell death
(apoptosis)
Pattern Formation
Differentiation is carefully orchestrated:
• Proliferation
• Cell migration
• Interactions (Induction)
• Epithelial-mesenchymal transformations
• Epithelial folding, movement,
evagination, fusion
• Apoptosis …
Apoptosis
• Lineage analysis of C.
elegans highlights
another outcome of cell
signaling, programmed
cell death or
apoptosis.
– The timely suicide
of cells occurs
exactly 131 times in
the course of C.
elegans’s normal
development.
– At precisely the
same points in
development,
signals trigger the
activation of a
cascade of “suicide”
proteins in the cells
destined to die.
Organism
Caenorhabditis elegans
Mammalian Cell
Inhibitor:
CED-9
Activator:
CED-4
Apaf1
Apoptotic
Protease:
CED-3
Caspase-8 or -9
Apoptosis
Apoptosis
Inhibition
Activation
a.
Inhibitor
b.
Bcl-2
If it weren’t for apoptosis, you may
look a little different…
• Apoptosis pathways in humans and other mammals are
more complicated.
• Research on mammals have revealed a prominent role for
mitochondria in apoptosis.
– Signals from apoptosis pathways or others somehow cause
the outer mitochondrial membrane to leak, releasing proteins
that promote apoptosis.
• A cell must make a life-or-death “decision” by somehow
integrating both the “death” and “life” (growth factor)
signals that it receives.
• Apoptosis is essential to the development of animal
morphogenesis (prevents webbing between fingers and
toes).
Model Organism (1)
The Worm
• The nematode C. elegans normally lives in the soil but is
easily grown in petri dishes.
– Only a millimeter long, it has a simple, transparent body with
only a few cell types and grows from zygote to mature adult in
three and a half days.
– Its genome has been sequenced.
– Because individuals are hermaphrodites, it is easy to detect
recessive mutations.
• Self-fertilization of heterozygotes will produce some homozygous recessive
offspring with mutant phenotypes.
• A further important feature is that every adult C. elegans
have exactly 959 somatic cells.
– These arise from the zygote in virtually the same way for every
individual.
Studying Worms
•
•
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•
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C. elegans is a very useful model organism for investigating the roles of cell
signaling and induction in development.
The pathway from fertilized egg to adult nematode involves four larval stages (the
larvae look much like smaller versions of the adult) during which the vulva
develops.
Six cells present on the ventral surface of the second-stage larva gives the vulva.
A single cell in the embryonic gonad, the anchor cell, initiates a cascade of
signals that establishes the fate of the vulva precursor cells.
Vulva development in the nematode illustrates several important developmental
concepts:
– In the developing embryo, sequential inductions drive the formation of organs.
– The effect of an inducer can depend on its concentration.
– Inducers produce their effects via signal-transduction pathways similar to
those operating in adult cells.
– The induced cell’s response is often the activation (or inactivation) of genes
which establishes the pattern of gene activity characteristic of a particular cell
type.
– Genetics as a powerful approach for elucidating the mechanisms of
development.
A fate map traces the development
of an embryo.
Model Organism (2)
The Fruit Fly
• The fruit fly Drosophila melanogaster was first chosen as
a model organism by geneticist T.H. Morgan and
intensively studied by generations of geneticists.
– The fruit fly is small and easily grown in the laboratory.
– It has a generation time of only two weeks and produces
many offspring.
– Embryos develop outside the mother’s body.
– In addition, there are vast amounts of information on its
genes and other aspects of its biology.
– However, because first rounds of mitosis occurs without
cytokinesis, parts of its development are superficially quite
different from what is seen in other organisms.
Sabotaging the Fruit Flies
•
•
•
•
In the 1940s, Edward B. Lewis demonstrated that the study of mutants could be
used to investigate Drosophila development.
– He studied bizarre developmental mutations and located the mutations on the
fly’s genetic map.
– This research provided the first concrete evidence that genes somehow direct
the developmental process.
In the late 1970s, Christiane Nüsslein-Volhard and Eric Weischaus pushed the
understanding of early pattern formation to the molecular level.
Their goal was to identify all the genes that affect segmentation in Drosophila.
– Mutations that affect segmentation are likely to be embryonic lethals, leading
to death at the embryonic or larval stage.
– Because of maternal effects on axis formation in the egg, they also needed to
study maternal genes.
After a year of hard work, they identified 1,200 (out of a fruit flies 13,000) genes
essential for embryonic development
– About 120 of these were essential for pattern formation leading to normal segmentation. They
were able to map these genes.
•
The results of detailed anatomical observations of development in several species
and experimental manipulations of embryonic tissues laid the groundwork for
understanding the mechanisms of development.
Life cycle of the fruit fly
Homeotic-Selector
HOX Genes
Background
Among the most fascinating kinds of abnormalities in animals are
those in which one normal body part is replaced by another.
William Bateson (1894) catalogued several oddities of this nature
coining the term homeotic to describe them.
Calvin Bridges (1915) isolated a spontaneous mutant of Drosophila
in which part of the haltere was transformed into wing tissue. The
mutant was called bithorax (duplication of a thoracic segment).
In the following decades other mutations affecting segment
identity were identified e.g. certain Antennapedia mutations
causing transformation of antennae into legs.
In Drosophila, genes controlling segment identity were found to
be transcription factors and coined to be homeotic-selector
genes, meaning that the fate of a given segment was selected by
the expression of specific homeotic genes.
Homeobox
• All homeotic genes of
Drosophila include a
180-nucleotide
sequence called the
homeobox, which
specifies a 60-aminoacid homeodomain.
– An identical or very
similar sequence of
nucleotides (often
called Hox genes) are
found in many other
animals, including
humans.
How Homeodomains Work
• Proteins with homeodomains probably regulate
development by coordinating the transcription of
batteries of developmental genes.
– In Drosophila, different
combinations of
homeobox genes are
active in different parts
of the embryo and at
different times, leading
to pattern formation.
Action of Signaling molecules
Paracrine factors
• Inducing factors controlling forms of
developing organ
• Mitogen regulating cell proliferation
• Morphogen acting in a dose-dependent
way to pattern the cell fates within a
target field
Morphogens
• Product of the mother’s bicoid gene is essential for
setting up the anterior end of the fly.
• The gene’s products are concentrated at the future
anterior end.
• This is a specific version of a general gradient
hypothesis, in which gradients of morphogens establish
an embryo’s axes and other features.
• The bicoid protein and other morphogens are
transcription factors that regulate the activity of some of
the embryo’s own genes.
• Gradients of these morphogens bring about regional
differences in the expression of segmentation genes,
the genes that direct the actual formation of segments
after the embryo’s major axes are defined.
Homeotic Mutations
The direction of homeotic transformations depends on whether the
mutation causes loss of homeotic gene function where the gene
normally acts or gain of function where the gene normally does not
act.
Ultrabithorax (Ubx) acts in the haltere to promote haltere
development and repress wing development. Loss of function
mutations in Ubx transform the haltere into a wing.
Dominant mutations that cause Ubx to gain function in the wing
transform that structure into a haltere.
In antenna-to-leg transformations of Antennapedia the mutants
reflect a dominant gain of Antennapedia gene function in the
antennae.
Examples of Homeotic Mutations
Normal adult fly
Antennapedia
mutant
Bithorax mutant
Homeotic-Selector Genes
 Homeotic gene products regulate development within
parasegmental domains. At this stage each band of cells along the
A-P axis express a unique combination of transcription factors
which control subsequent cell development.
 Patterns of selector gene expression are determined in the early
embryo and maintained into the adult fly. Continuous expression is
required to specify structures along the A-P axis.
 The genes are ordered on the chromosome in the order of their
expression with respect to the parasegments of the Drosophila
embryo.
 Two of the best studied selector genes are Antennapaedia (Antp)
which dominates development of the fourth parasegment and
Ultrabithorax (Ubx) which largely controls the sixth parasegment.
Homeotic-Selector Genes
 Transcription of the Antp and Ubx genes begins in the third hour of
embryogenesis in domains determined in part by various gap-gene
products with boundaries set up by the segmentation genes.
 Homeotic genes act in a cell autonomous manner – a cell will
behave according to homeotic gene expression of its own
regardless of the pattern of expression of its neighbours
 Maintenance of homeotic gene expression is through self
regulation and the Polycomb-like class of genes.
 Selector genes specify epidermal structures – the musculature,
neural tissue and gut along the A-P axis.
 Two homeotic selector gene clusters occur in two groups on the
same chromosome – five (the Antennapedia Complex – ANT-C)
are expressed in the head region while genes in the Bithorax
Complex (BX-C) are mainly expressed in thorax or abdominal
regions.
Eight Genes Regulate the Identity of Within
the Adult and Embryo
labial (lab)
proboscipedia (pb)
Deformed (Dfd)
Sex combs reduced (Scr)
Antennapedia (Antp)
Ultrabithorax (Ubx)
abdominal A (abd-A)
Abdominal B (Abd-B)
Homeotic-Selector Genes
 Homeotic genes encode nuclear proteins containing a DNAbinding motif called a homeodomain.
 The products are transcription factors that specify segment identity
by activating multiple gene expression events.
 The genes are initially activated imprecisely by the concentration
gradients of gap gene products.
e.g. Ubx is switched on between certain concentrations of
hunchback to give a broad band of expression near the middle of
the embryo. Later, fushi tarazu and even skipped sharpen the
limits of Ubx expression which comes into register with the anterior
boundaries of specific parasegments.
 The BX-C and ANT-C genes have extensive non-coding
sequences (introns) that are critical in regulating their individual
expression.
Fate map of a Drosophila embryo at the cellular blastoderm stage.
• The embryo is shown in side view and in cross-section, displaying the relationship between the
dorsoventral subdivision into future major tissue types and the anteroposterior pattern of future
segments.
• A heavy line encloses the region that will form segmental structures.
• During gastrulation the cells along the ventral midline invaginate to form mesoderm, while the cells
fated to form the gut invaginate near each end of the embryo.
• Thus, with respect to their role in gut formation, the opposite ends of the embryo, although far apart
in space, are close in function and in final fate.
Patterns of
Expression
The patterns of expression compared to
the chromosomal locations of the
genes of the HOM complex.
•
The sequence of genes in each of the
two subdivisions of the chromosomal
complex corresponds to the spatial
sequence in which the genes are
expressed.
•
Note that most of the genes are
expressed at a high level throughout one
parasegment (dark color) and at a lower
level in some adjacent parasegments
(medium color) where the presence of
the transcripts is necessary for a normal
phenotype, light color where it is not).
•
In regions where the expression domains
overlap, it is usually the most "posterior"
of the locally active genes that
determines the local phenotype.
•
The drawings in the lower part of the
figure represent the gene expression
patterns in embryos at the extended
germ band stage, about 5 hours after
fertilization.
Effects of Mutations
in Bithorax Complex
Contribution of BX-C genes — Ubx, abdA,
and AbdB—to determination of
parasegment identity.
• The numbers above each larva indicate the
parasegments; those below, the corresponding
segments.
• The cuticular pattern of larvae is used to
assign an identity to each parasegment (PS),
which is indicated by color, as depicted in the
wild type at the top.
• Red PS and segment labels indicate
abnormal patterns that do not correspond
exactly to any found in wild-type larvae.
Expression and lethal embryonic
phenotypes of homeotic genes.
This phenotypic analysis has
been confirmed by the expression patterns
of the various genes in various mutant
backgrounds.
The anterior limits of the domain
of expression of a particular homeotic
gene are presumably set by a
collaboration between gap genes and pairrule genes.
Removal of an anteriorly-acting
homeotic has no effect on expression or
phenotype in the domain of a more
posteriorly acting homeotic gene.
Antp expands posteriorly from PS4 to
PS6 in a Ubx mutant
Antp expands posteriorly from PS4 to
PS12 in a Ubx AbdA mutant
Antp expands posteriorly from PS4 to
PS14 in a Ubx AbdA AbdB mutant
Ubx expands posteriorly from PS6 to
PS12 in a AbdA mutant
Ubx expands posteriorly from PS6 to
PS14 in a AbdA AbdB mutant
Gain- and Loss-of-Function Studies
 Conservation of order of homeotic gene expression plays an
important role in controlling patterning.
 “Out of order” expression in single mutants lacking abd-A causes
marked defects in parasegments 10-14 and they do not correspond
to any wild-type parasegment. Therefore Ubx and Abd-B do not
provide recognisable patterning information in the absence of abd-A.
 For gain of function, transfected Ubx can be induced to be uniformly
expressed along the A-P axis (as opposed to parasegments 5 and 6).
In this scenario parasegments 6 to 14 form normally but
parasegments 1 to 5 are transformed into parasegment 6.
Gain- and loss-of-function studies show a consistent relation
between selector genes: Genes expressed more posteriorly
suppress the action of genes expressed more anteriorly.
Binding Sites of Hox Proteins
• Hox proteins bind DNA sequences – but
different Hox proteins bind with high affinity to
the same short DNA sequences found once
every kilobase.
• The ability of Hox proteins to control
expression of different genes depends on the
product of the extradenticle (exd) gene.
• Exd binds different Hox proteins forming
different heterodimers that bind selectively to
specific target sequences
• Exd may also contribute to specificity of Hox
function by converting bound Hox proteins from
repressors to activators.
• Exd may also operate independently to
repress other genes.
Role of Exd protein in conferring DNA-binding specificity on Drosophila Hox proteins.
(a) Various Hox proteins, including Dfd and Lab, bind to a 10-bp consensus sequence that differs in the nucleotides (N) at
the two central positions.
(b) (b) Exd-Dfd and Exd-Lab heterodimers specifically recognize Hox-binding sites in which the central dinucleotide is
TA or GG, respectively.
Maintaining Hox Gene Expression
 The transcription-control regions of some Hox genes contain binding
sites for their encoded proteins – autoregulatory loop (e.g. lab and
Dfd).
 A second mechanism requires proteins that modulate chromatin
structure. There are two classes – the trithorax group and the
polycomb group.
 Early patterning requires repression as well as activation of gene
expression. Polycomb proteins have a repressive effect on the
expression of Hox genes.
 Polycomb proteins bind multiple chromosomal locations to form
large macromolecular complexes and this becomes “locked in”.
 Trithorax proteins maintain the expression of many Hox genes.
These also form large multiprotein complexes at multiple
chromosomal sites, but mainain an open chromatin structure and
stimulate gene expression.
Pattern of Binding of Polycomb Protein
The normal pattern of binding of Polycomb protein to Drosophila giant chromosomes, visualized with an
antibody against Polycomb.
The protein is bound to the Antennapedia complex (ANT-C) and the bithorax complex (BX-C) as well as
about 60 other sites.
Conservation of Homeotic-Selector Genes
 The organisation of genes within ANT-C and BX-C are similar to
mammalian homologues occur in four gene clusters located on
different chromosomes (Hox complex).
 Current thinking on the evolution is as follows:
A primordial homeotic selector gene of a common ancestor of
worms, flies and vertebrates underwent repeated duplication to
form a series of genes in tandem.
In the Drosophila sublineage this single complex became split
into separate Antennapedia and bithorax complexes.
In the lineage leading to the mammals the whole complex was
repeatedly duplicated to give the four Hox complexes.
The parallelism between mammals and Drosophila is not perfect
as some individual genes have been duplicated and others lost
since the complexes diverged.
Comparison of Drosophila and Human Genes
Comparison of Drosophila ANT-C and BX-C genes and the four human Hox complexes.
For purposes of alignment, the ANT-C and BX-C (top) are shown adjacent to each other, although they are separated on chromosome III in
the fly genome. Each Hox complex (HoxA – HoxD) can be divided into 13 regions numbered 1 – 13.
Genes in different Hox complexes with the same number are homologs called paralogs (e.g., HoxA1 and HoxB1). Empty boxes indicate that
the corresponding genes have not been identified. Similarities in expression patterns and sequences strongly suggest that the fly labial (lab),
proboscipedia (pb), Deformed (Dfd), and Abdominal B (AbdB) genes are analogous to regions 1, 2, 4, and 9, respectively.
The set of fly genes including Sex combs reduced (Scr), Antennapedia (Antp), Ultrabithorax (Ubx), and abdominal A (abdA) (purple) is
similar to regions 5 – 8, but precise correlations between the fly genes and Hox regions are not possible at present.
Horizontal arrows indicate direction of transcription. Anterior and posterior refer to the order of expression of these genes along the body
axis.
Homeodomains of Drosophila Hom Genes
Similarities of Drosophila and Vertebrate Hox Proteins
Many Developmental Regulatory Genes
are Transcription factors
Classes of transcription factors:
Homeodomain proteins (Hox)
POU
Basic helix-loop-helix
Basic leucine zipper
Zinc finger
Nuclear hormone receptors
DNA-bending proteins
Associated phenotypes:
Androgen receptor
AZF1
CBFA1
CSX
EMX2
Estrogen receptor
Androgen insensitivity syndrome
Azoospermia
Cleidocranial dysplasia
Heart defects
Schizencephaly
Growth reg. problems, …
Transcription Factors Associated
phenotypes and diseases
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Forkhead-like 15
Gl13
HOXA-13
HOXD-13
LMXIB
MITF
Pax2
Androgen receptor
AZF1
CBFA1
CSX
EMX2
Estrogen receptor
PAX3
PAX6
PTX2
PITX3
POU3F4
SOX9
SRY
TBX3
TBX5
TCOF
TWIST
WTI
Thyroid agenesis, cleft palate
Grieg syndrome
Hand-foot-genital syndrome
Polysyndactyly
Nail-patella syndrome
Wardenburg syndrome type 2
Renal-coloboma syndrome
Androgen insensitivity syndrome
Azoospermia
Cleidocranial dysplasia
Heart defects
Schizencephaly
Growth reg. problems, …
Waardenburg syndrome type 1
Aniridia
Reiger syndrome
Congenital cataracts
Deafness and dystonia
Campomelic dysplasia, male sex reversal
Male sex reversal
Schinzel syndrome (ulna-mammary syndrome)
Holt-Oram syndrome
Treacher-Collins syndrome
Seathre-Chotzen syndrome
Urogenital anomalies
Model Organism (3)
• A vertebrate model, the zebrafish Danio rerio, has
some unique advantages.
– These small fish (2 - 4 cm long) are easy to breed in the
laboratory in large numbers.
– The transparent embryos develop outside the mother’s
body.
– Although generation time is two to four months, the early
stages of development proceed quickly.
• By 24 hours after fertilization, most tissues and early
versions of the organs have formed.
• After two days, the fish hatches out of the egg case.
– The study of the zebrafish genome and related phenotypes
is an active area.
Model Organism (4)
The Mouse
• The most economic mammals with high reproduction
efficiency, strong immune system and small size.
• Over hundreds of inbred, outbred and mutant with clear
genetics and phenotypes.
• Gestation: 19-21days
• Breast feeding：4 weeks
• Litter size: 6-14
• Sex mature：male 6-8 weeks，female 4-6 weeks
• Estrus：as short as 5 days，male always ready for mating
• Mating time：Midnight, plug day considered E0.5
• Chromosomes: 20 pairs，haploid 2.71 billion bp，0.3pg
DNA
• Body weight average 30-40g，blood 2ml, heart beat 500700bpm
Terms
• Outbred：Offspring of different strains for better
survival and reproduction
• Inbred: >20 brother-sister mating, no lethal genes
but weak in survival and reproduction
• F1：First generation of 2 inbred strain,
• Isogenic: Strains with same genetic background
• Coisogenic: Mutant in pure genetic background
• Congenic: For gene locus mapping between 2
inbred
• Cosmic: One chromosomal change
Popular Strains
• Inbred
– DBA, diluted brown, nonagouti, first inbred ever, made by
Clarence Cook Little in 1907-1950
– A strain, from Bagg Albino and an albino from Clarence Cook
Little, hig mammary and lung tumor
– C3H, CBA, C, CHI, and C12I , all derived from mating of Bagg
Albino with DBA
– C57BL and C57BR from Miss A.E.C. Lathrop’s female 57 and
male 52
– BALB/c, Bagg Albino
– 129sv/J
• Outbred
– CD1
– Kunming
Types of crossing
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Matings of like homozygotes, +/+ x +/+
and a/a x a/a, called incrosses
Matings of unlike homozygotes +/+ x a/a
and a/a x +/+, called crosses
Matings of a homozygote and a
heterozygote, +/+ x a/a, a/+ x +/+, a/+ x
a/a, and a/a x a/+, called backcrosses
Matings of heterozygotes, a/+ x a/+
called intercrosses
Introduction
• The mouse has always been a good
embryological model, generating easily and
quickly.
• Mouse embryology really expanded when
molecular biologists used mice for gene
knockouts, necessary to understand the effect of
an unknown gene in the development.
• There are over 450 different strains of inbred
research mice.
Mouse Embryonic Development
Live cell imaging and tracking
• (A) GFP-GPI expression from
E.2.5 to E4.5. Deconvolved
fluorescence and differential
interference contrast time-lapse
images.
• (B) Embryos stained to reveal
Gata4-positive cells adjacent to
mature blastocyst cavity
confirming normal development
during each imaging session.
Gata4-positive cells were present
in a one-cell-thick surface layer.
• (C) Lineage tree from
representative embryo. All cells
were traced to the early 32-cell
blastocyst; then inside cells were
traced to late blastocyst.
Allocation to trophectoderm (TE),
EPI, or PE and apoptosis (A) are
indicated.
Cell fate decisions and axis determination in
the early mouse embryo
• The early cell fate decisions lead to the
generation of three lineages in the preimplantation embryo: the epiblast, the
primitive endoderm and the trophectoderm.
• Shortly after implantation, the anteriorposterior axis is firmly established
Placenta Development
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placenta originates from the
ectoplacental cone and the extraembryonic ectoderm.
Endothelial cells derive from the
allantois.
embryonic day (E 10) - placenta
divided into three layers associated
with maternal decidual cells.
Labyrinth - (equivalent to human
villi) a selective barrier on the fetal
side, is an array of fetal and
maternal vessels.
Junctional zone - (spongy layer)
produce hormones and contains
numerous cavities. The trophoblast
cells form spongiotrophoblasts and
the glycogen cells, that later (E 12.5)
migrate into the maternal decidua.
Giant cells - next to the uterine cells
form the outermost fetal cell layer
until (E 12).
Mouse Placenta Vasculature (E16.5)
Making Muscles
Transgenic Studies
• Put genes back into the genome and
watch for function
• Trace the developmental cells for their fate
using fluorescent proteins
• Genetic disease models
Mouse Knockouts
• Knowledge about mouse development has rapidly expanded as
genetic "knock out " studies becomes available.
• This is the ideal system to precisely decipher the gene function.
• This technology uses ES cell culture, homologous recombination in
mouse genome and embryo manipulation.
• The good old fashioned histology is most useful.
• With the genomic sequence available and discovery of many unknown
genes, gene knock out becomes the first of choice.
• There is a database of all existing mouse knockouts and their
consequences.
• Gene targeting in embryonic stem cells has become the principal
technology for manipulation of the mouse genome, offering unrivalled
accuracy in allele design and access to conditional mutagenesis.
• To bring these advantages to the wider research community, largescale mouse knockout programmes are producing a permanent
resource of targeted mutations in all protein-coding genes.
A conditional knockout resource for the genome-wide
study of mouse gene function
• A high-throughput gene-targeting pipeline has been
established for the generation of reporter-tagged,
conditional alleles.
• Computational allele design, 96-well modular vector
construction and high-efficiency gene-targeting strategies
have been combined to mutate genes on an
unprecedented scale.
• So far, more than 12,000 vectors and 9,000 conditional
targeted alleles have been produced in highly germlinecompetent C57BL/6N embryonic stem cells.
• High-throughput genome engineering highlighted by this
study is broadly applicable to rat and human stem cells
and provides a foundation for future genome-wide efforts
aimed at deciphering the function of all genes encoded by
the mammalian genome."
More than just Knockout
• Recent progress in high throughput
sequencing, mouse genome study, animal
cloning and induced pluripotent stem cells
have pushed gene to a new level.
Animal Cloning
Stem Cells
Stem cells have the potency to differentiate into multiple cell types.
They can differentiate into multiple types of cells and are multipotent or
any types of cells (pluripotent).
They continue to reproduce themselves under appropriate
conditions and differentiate into specialized cell types under
selective conditions.
The adult body has various kinds of stem cells to replenish
specialized cells or damaged cells.
For example, stem cells in the bone marrow give rise to all the
different kinds of blood cells.
A recent surprising discovery is the presence of stem cells in the
brain that can continues to produce certain kinds of nerve cells.
Scientists are learning to identify and isolate these cells from various
tissues to culture them.
– Stem cells from early embryos are somewhat easier to culture than
those from adults and can produce differentiated cells of any type.
– These embryonic stem cells are “immortal” because of the presence
of telomerase that allows these cells to divide indefinitely.
The Nobel Prize in Physiology or Medicine 2012
Sir John B. Gurdon, Shinya Yamanaka
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In plants, one live cell can remain totipotent,
the potential to form the mature organism.
Cloning mammals using cultured cells
Gurdon worked out that cells could be
reprogrammed into a more immature state
in 1962
In 2006, Yamanaka worked out how to turn
mature cells into stem cells by introducing a
few genes. Yamanaka's 'induced pluripotent
stem cells' (iPS) removed the need to use
live human embryos to create versatile stem
cells
Induced Stem Cells (iPS)
OCT4, SOX2, Klf4 and c-Myc or Oct4 only
can induce stem cell reprogramming
Under the right conditions, cultured stem cells
can differentiate into specialized cells
Push the development
backward and redevelop
A high-resolution anatomical atlas
of the transcriptome in the mouse embryo
• Generation of anatomy-based expression
profiles for over 18,000 coding genes and
over 400 microRNAs.
• Identification of 1,002 tissue-specific
genes that are a source of novel tissuespecific markers for 37 different
anatomical structures
• Transcriptional mapping for development
Summary
• Master developmental regulation involves
chromosomal remodeling, up- and down- regulating
transcription factors and massive gene reprogramming.
• Growth factors, Signaling molecules are mediators of
the complex processes.
• During the development, all the genes involved are
regulated by their position and timing.
Homeostasis
Proliferation
Differentiation