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Animal Development
Lecture 5
Model Systems
1
Model Animals
•1.2 millions of animal species on earth.
•Reasons: historical reason, ease of study
and biological interest
•A few have been studied extensively;
each has advantages and
disadvantages.
2
•Xenopus laevis: development is independent but
poor genetics.
•Chick: available, surgical manipulation and in
vitro culture but poor genetics.
•Mouse: good genetics but development is in
utero.
•Drosophila: great genetics, great development
(recent Nobel Prize to Lewis,
Nusslein-Volhard & Wiechaus).
•C. elegans: has less than 1000 cells and is
transparent.
•Arabidopsis thaliana: flowering plant.
3
Model Animals
•only 4 species are intensively studied as
models
invertebrates:
Fruit fly (Drosophila melanogaster)
Nematode (Caenorhabditis elegans )
vertebrates:
Mouse (Mus musculus)
Zebrafish (Danio rerio)
4
The skeleton of a mouse
embryo illustrates the
vertebrate body plan
1 mm
1 mm
The unfertilized
egg of Xenopus
5
implanted
Vertebrate embryos go through a similar phylotypic stage, but the
embryos show considerable differences in form before gastrulation
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The phylotypic stage
• At the end of gastrulation all embryos appear to be
similar (the phylotypic stage).
• Structures that are common to the phylotypic stage of
the vertebrates are:
1) the notochord (an early mesoderm structure along A/P
axis),
2) the somites (blocks of mesoderm on either side of
notochord which form the muscles of the trunk & limbs),
3) the neural tube - ectoderm above notochord forms a
tube (brain and spinal cord).
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Life cycle of the Xenopus laevis
0.5 mm
1 mm
1 cm
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Polar Body Formation
9
Cleavage of Xenopus Embryo
Gastrulation in Amphibians
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Xenopus laevis : fertilization and early growth
1. one sperm enters animal region
2. completes meiosis
3. egg and sperm nuclei fuse
4. vitelline membrane lifts
5. yolk rotates down (15 minutes)
6. cortical rotation occurs (60 minutes)
7. 1st cleavage occurs (90 mins) A/V
8. 2nd cleavage (110 mins) A/V 90 degrees to 1st
9. 3rd cleavage (130 mins) equatorial (4 small
animal and 4 large vegetal= 8 blastomeres)
11
Xenopus laevis: blastulation & gastrulation
• The blastula (after 12 divisions) has radial symmetry.
• The marginal zone will become mesoderm and
endoderm.
• Internalization of the mesoderm and endoderm starts at
the blastopore.
1)
2)
3)
4)
5)
Mesoderm and endoderm converge and begin to
move inwards at dorsal lip of the blastopore.
Mesoderm and endoderm extend in along A/P axis.
Ectoderm spreads to cover embryo (epiboly).
Dorsal endoderm separates mesoderm from the
space between the yolk cells, the archenteron (future
gut).
Lateral mesoderm spread to cover inside of
archenteron.
12
Xenopus laevis: late gastrulation
•By the end of gastrulation:
1) dorsal mesoderm is beneath dorsal ectoderm,
2) mesoderm spread to cover gut,
3) epiboly - ectoderm covers embryo ,
4) yolk cells are internalized (food source), and
5) dorsal mesoderm develops into
a) notochord (rod along dorsal midline) and
b) somites (segmented blocks of mesoderm
along notochord).
13
Neurulation
in
Amphibians
14
0.2 mm
A cross-section through a stage 22 Xenopus embryo
just after gastrulation and neurulation are completed
15
The early tailbud stage
(stage 26) of a Xenopus
embryo
1 mm
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17
Xenopus laevis: Neurulation
• Neuralation or neural tube formation:
1) The neural plate is the ectoderm located
above notochord and somites.
2) The edge of the neural plate forms neural folds
which rise towards midline.
3) The folds fuse to form neural tube.
4) The neural tube sinks below epidermis.
• The anterior neural tube becomes brain. Mid
and posterior neural tube becomes spinal
cord.
18
Xenopus laevis: Somites
•The somites:
-
The dorsal part of somites become dermatome
(future dermis).
The rest of each somite becomes vertebrae
and trunk muscles (and limbs).
•Lateral plate mesoderm becomes heart, kidney,
gonads and gut muscles.
•Ventral mesoderm becomes blood-forming
tissues.
•Also at this stage, the endoderm gives rise to the
lining of the gut, liver & lungs.
19
Xenopus laevis: tail bud stage
•After gastrulation comes the early tail bud
stage
•In the anterior embryo:
a) the brain is divided,
b) eyes and ears form,
c) 3 branchial arches form (anterior arch later
becomes the jaw).
•In the posterior embryo, the tail is formed last
from dorsal lip of blastopore by extension of
notochord, somites and neural tube.
20
Xenopus laevis : neural crest cells
•Neural crest cells come from the edges of the
neural folds after neural tube fusion.
•Neural crest cells detach and migrate as
single cells between the mesodermal tissues
to become:
1) sensory and autonomic nervous systems
2) skull
3) pigment cells
4) cartilage
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The development of a hen egg at the time of laying
22
Life cycle of the chicken
1 mm
1 mm
50-53 hpf
10 mm
8-9 D pl
23
Cleavage and
epiblast
formation in the
chick embryo
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Chick embryo: the blastodisc
•The blastodisc arises through cleavage (20 h).
•The blastodisc can be divided into two areas:
1) the area pellucida (a light area) surrounded by
2) the area opaca (a dark ring).
•The posterior marginal zone forms at the junction of
the area pellucida and the area opaca and defines
the dorsal side and posterior end of the embryo.
•The hypoblast (the source of extra-embryonic
tissues) develops as a layer on top of yolk and
develops from cells from the posterior marginal layer
and the overlying cells of the blastoderm.
25
Anterior
Ingression of mesoderm and endoderm
during gastrulation in the chick embryo
26
Regression of Hensen’
s node
Head fold and notochord formation during
node regression in the chick embryo
27
Chick embryo: the primitive streak
•The primitive streak is a slit or line on the disc which
lays down the A/P axis.
•This structure begins to form from the posterior
marginal zone and extends to a point in the central
region of the disc.
•Cells move towards the streak, and mesoderm and
endoderm internalize at this site.
•When the primitive streak reaches its greatest length,
the anterior end begins to regress back to the
posterior end.
•The anterior end of the regressing streak is known as
Hensen's Node.
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Chick embryo: gastrulation
•As Hensen's Node moves toward the posterior,
several structures form behind it:
1)
2)
3)
The head fold (from ectoderm and endoderm)
The notochord and somites (from mesoderm)
The neural tube forms above the notochord (from
ectoderm)
•(The anterior structures are formed first while the
posterior structures are completed last.)
4) Neural folds fuse at the dorsal midline and neural
crest cells migrate away
5) The head fold separate, gut forms and heart pieces
fuse to form heart.
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0.1 mm
Scanning electron
micrograph of chick early
somites and neural tubes
Development of the
neural tube and
mesoderm in the chick
embryo
30
Development of the chick embryo
Hensen’
s node
1 mm
13-somite stage
1 mm
20-somite stage
1 mm
40-somite stage
31
The
development
of chick
embryo
- 24 hpl
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The
development
of chick
embryo
- 33 hpl
33
The development of chick embryo - 48 hpl
34
The development of chick
embryo - 72 hpl
35
The development of chick
embryo - 96 hpl
36
The extra-embryonic structures and
circulation of the chick embryo
37
Life cycle of the mouse
Cleavage and
blastulation (0-5 days)
10 m
100 m
Organogenesis (10-14 days) /
Fetal growth and
development (14-19 days)
Implantation, gastrulation and
early organogenesis (5-10 days)
1 mm
Fertilized egg
8 D pf
14 D pf
38
Cleavage in the Mouse Embryo
Early Post-implantation Development of the Mouse Embryo 39
Mouse embryo: fertilization
•Fertilization occurs in oviduct.
•Cleavage occurs in oviduct: 1st at 24 hours and
every 12 hours after that to form the morula (a ball
of cells).
•Blastomere compaction happens at 8 cell stage.
•Smooth inner membranes and outer membranes
are covered with microvilli.
•Trophectoderm: becomes extra-embryonic
tissues.
•Inner cell mass (ICM): becomes the embryo plus
some extra-embryonic tissues.
40
Mouse embryo: blastocyst
•The morula (~32 cell stage) has 2 cell fates: 1) inner
8 cells (Inner Cell Mass) and 2) outer ~20 cells
(trophectoderm).
•In the blastocyst (~3½ days), the trophectoderm
and ICM are established.
•Fluid is pumped in to expand cavity and increase
the size of the blastocyst.
•blastocyst: preimplantation (3½ - 4½ days)
•The surface of ICM will become the primitive
endoderm while the remaining becomes primitive
ectoderm (= epiblast).
•Implantation occurs. The zona pellucida is
discarded and blastocyst attaches to uterine wall.
41
Gastrulation in the Mouse Embryo
42
Early Post-implantation Development of the
Mouse Embryo
43
Turning in the Mouse Embryo
44
Mouse embryo: post-implantation
•In the first two days post-implantation, the mural
trophectoderm (cells that are not in contact with the
ICM) gives rise to polyploid trophoblast giant cells.
•The rest of trophectoderm becomes the
ectoplacental cone and the extra-embryonic
ectoderm which give rise to the placenta.
•Primitive endoderm migrates:
1)
2)
to cover inner surface of mural trophectoderm to
become the parietal endoderm and
to cover egg cylinder and epiblast to become the
viseral endoderm
•Six days after fertilization, the epiblast is cupshaped.
45
Mouse embryo: gastrulation
• 6½ days after fertilization:
• The primitive streak forms at the start of gastrulation at
the future posterior end. (Inside cup is future dorsal side)
• Cells move through the streak and spread forward and
laterally between the ectoderm and the visceral
endoderm to form the mesoderm.
• Later, the definitive endoderm (from epiblast) will
replace the visceral endoderm.
• The primitive steak first elongates, then at the anterior tip
of the primitive streak, the node forms.
• Then notochord and somites form anterior to the node.
• Cells migrate through mesoderm to form endoderm
(gut).
46
Mouse embryo: late embryogenesis
•By 8½ days after fertilization,
1) the neural folds form at anterior and dorsal,
and
2) the embryonic endoderm internalizes to
form the gut.
•9 days after fertilization embryogenesis is
complete.
47
Originate
from India
0.5 mm
0.5 mm
Sphere
stage
14-somite
stage
1 cm
Life cycle of the zebrafish
48
The Development
of Zebrafish
0.75
h
1h
1.25 h
1.5 h
1.75 h
2h
2.5 h
3.3 h
3.5 h
3.8 h
4.3 h
4.7 h
10 min
40 min
Bar = 250 µm
49
Cleavage of the zebrafish embryo is initially
confined to the animal (top) half of the embryo
Epiboly and Gastrulation in the Zebrafish
50
24 h
28 h
28 h
33 h
33 h
36 h
36 h
42 h
42 h
120 h
48h
48 h
60 h
60 h
72 h
72 h
120 h
51
Why Use Zebrafish As A Model To Study Human
Disease
1. Zebrafish genome is much smaller than the human.
a.
Genome about 1.7x109 bp
b.
Only 10% of the genome is used for gene expression, majority of
the rest genome is composed of sequence repeat.
2. Excellent for embryological, developmental and genetic
studies:
a.
b.
c.
d.
Availability of a large number of eggs.
Rapid external development and transparency during most of
the embryogenesis.
Short generation time: 3 months
Maintain and care is considerably easy.
3. The generation of zebrafish mutant phenotypes do
resemble the human disease in certain condition.
4. Maternal effect function can be studied through the
zebrafish system.
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0.1 mm
0.1 mm
0.1 mm
Life cycle of the Drosophila melanogaster
53
Cleavage of the Drosophila Embryo
Gastrulation
in
Drosophila
54
0.1 mm
Gastrulation, germ band extension, and
segmentation in the Drosophila Embryo
Ventral
view of a
0.1 mm
Drosophil
larva
Hatch
55
Imaginal discs
give rise to
adult structures
at
metamorphosis
56
57
10 m
10 m
0.5 m
Life cycle of the Caenorhabditis elegans
58
Cleavage of the C. elegans Embryo
Cell lineage and cell fate in
the early C. elegans
Embryo
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Total - 959 cells
Caenorhabditis elegans Embryo larva at the L1 stage (20 hpf)
60
Identification of developmentally important genes
• The developmental genetics of Drosophila and mice
are best known.
• Homologous genes identified in these organisms are
found in other species.
• Dominant (or semi-dominant) mutations: one copy of
mutant gene produces mutant state.
• Recessive mutations: two copies of a mutant gene
gives the mutant state.
• Mutants can arise spontaneously but induced mutation
and screening has become the standard way to
identify developmentally important genes.
61
Types of Mutations
62
Genetics of the semi-dominant mutation
Brachyury (T) in the mouse
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Genetic
screening to
produce
homozygous
mutant
zebrafish
embryos
64
Mutagenesis
and genetic
screening
strategy for
identifying
developmenta
l mutants in
Drosophila
65