Download Chapter 7 Part 1 File

Amphibians & Fish
Early Development and Axis
Formation
Chapter 7 – Part 1
1
Amphibian and Fish
• Backbone of vertebrate embryology 
• Frogs (Xenopus)
– Large cells and rapid development
– But, long period before fertile
• Zebra fish (Danio rerio)
– Model organism for study of vertebrate
development
•
•
•
•
Breed each year
Easily maintained
Transparent embryos
At 24 hours after fertilization – embryo has
formed most of its organ primordia
2
Fertilization, Cortical Rotation, and
Cleavage
• Fertilization and cortical rotation
– Can occur anywhere in animal hemisphere of
amphibian
– Sperm entry point determines orientation of
the dorsal ventral axis
• Marks ventral side
• 180° opposite sperm entry point marks the
dorsal side
– Centrioles organize microtubules in egg
• Arrange in parallel array in vegetal cytoplasm
• Cortical cytoplasm Rotates 30° relative to
internal cytoplasm
• Array disappears when rotation stops
3
Fertilization, Cortical Rotation, and
Cleavage
7.1 Formation of the parallel arrays
of microtubules in the vegetal
hemisphere along the future
dorsal-ventral axis
A. 40% first cell cycle complete –
microtubules begin forming
B. 50% first cell cycle complete –
more microtubules, but lack
polarity
C. 70% first cell cycle complete –
parallel array of microtubules
4
Fertilization, Cortical Rotation, and
Cleavage
• Opposite sperm entry site
– Gray crescent
• Where gastrulation will begin
7.1 Reorganization of cytoplasm in the newly fertilized frog egg
5
Fertilization, Cortical Rotation, and
Cleavage
7.1 Reorganization of cytoplasm in the newly fertilized frog egg
6
Unequal Radial Holoblastic Cleavage
• Unequal radial holoblastic cleavage
– Radially symmetrical and holoblastic
– Contains more yolk
• Concentrated in vegetal pole
– 1st cleavage bisects gray crescent
– 2nd cleavage starts before 1st finishes
– Uneven division due to presence of yolk in
vegetal hemisphere
• Even with unequal size
• Blastomeres continue to divide at same rate
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Unequal Radial Holoblastic Cleavage
7.2 Cleavage of a frog egg
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Unequal Radial Holoblastic Cleavage
7.3 Scanning electron micrographs
of frog egg cleavage
A. First cleavage
B. Second cleavage (4cells)
C. Fourth cleavage (16 cells)
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Unequal Radial Holoblastic Cleavage
• Unequal radial holoblastic cleavage
(cont’)
– Small micromeres at animal pole
– Large macromeres at vegetal pole
– Regulated by MPF (mitosis promoting
factors)
– At 16-64 cell stage
• called MORULA (mulberry)
10
Unequal Radial Holoblastic Cleavage
• At 128 cell stage
– Blastocoel formed
– Embryo considered a
Blastula
– 2 functions:
• Permit cell migration during
gastrulation
• Prevent cells beneath it from
interacting prematurely with
cell above
– Roof of blastocoel in animal
hemisphere
• Animal cap
• If placed near bottom of
vegetal cells – cap cells
become mesoderm
Cleavage to Blastula
11
Unequal Radial Holoblastic Cleavage
– Mesoderm formed next to vegetal
endoderm precursors
– Animal cap destined to become nerves and
skin
– Cell adhesion molecules keep cells together
• EP cadherin
7.4 Depletion of EP-cadherin mRNA in the Xenopus oocyte
results in the loss of adhesion between blastomeres and the
obliteration of the blastocoel
12
Mid Blastula Transition: Preparing for
Gastrulation
• Very few genes transcribed during early
cleavage
– Nuclear genes are not activated until the 12th cell
cycle
• Acquires capacity to become motile
• Mid blastula transition (MBT)
– Egg factors initiate this (absorbed by newly made
chromatin)
– Different genes activated in different cells
– Cell cycle acquires gap phases
– Blastomeres acquire capacity to become motile
13
Amphibian Gastrulation
• Multiple ways of gastrulation in different
amphibians
• Xenopus fate map
– Goal is to bring inside embryo areas destined to
form endodermal organs
– Blastula has different fates depending on whether a
cell is located deep or superficially in the layers
• Mesoderm precursors exist mainly in deep layers
• Endoderm (bottom) and Ectoderm (top)
14
Vegetal Rotation and the Invagination
of the Bottle Cells
• Gastrulation movements in the frog
– To position the mesoderm between the
outer ectoderm and the inner endoderm
• Vegetal rotation and the invagination of
the bottle cells
– Gastrulation starts on the future dorsal side
of embryo
• Below equator
• Region of gray crescent
• Region opposite the sperm entry point
15
Amphibian Gastrulation
7.6 Cell movements during frog gastrulation
16
Vegetal Rotation and the Invagination
of the Bottle Cells
– At this point, cells invaginate to form a
slitlike blastopore called bottle cells
• Change shape
• Maintain contact with outside cells
– Bottle cell’s line the archenteron
• Similar to sea urchins except gastrulation in
frogs begins in marginal zone not ventral
region
• Endoderm not as large/yolky here as other
vegetal blastomeres
17
Vegetal Rotation and the Invagination
of the Bottle Cells
– Experiments (Hardin & Keller) proved:
• Dorsal lip cell removed placed on inner
endoderm
• Formed bottle cells and sank below the surface
– Once involution starts
• Gastrulation will proceed without bottle cells
– Coordinated involution of subsurface cells
more important
18
Vegetal Rotation and the Invagination
of the Bottle Cells
– Vegetal rotation
• Initiated by internalization of endoderm and
mesoderm
• 2 hours before bottle cells are formed
– Rotation puts prospective pharyngeal
endoderm adjacent to the blastocoel and
above involuting mesoderm
– Marginal cells form endodermal lining of
archenteron
– If the involuting marginal zone (IMZ)
cells were removed
• Archenteron formation would stop
19
Vegetal Rotation and the Invagination
of the Bottle Cells
7.8 Early movements of Xenopus gastrulation
20
Involution at the Blastopore Lip
• Involution of blastopore lip
– Involution of marginal zone cells
– Animal cells undergo Epiboly and converge
at blastopore
– Dorsal lip of blastopore
• Migrating Margin cells turn inward and travel
along inside animal hemisphere
• Form the lip of the blastopore
– Constantly changing
21
Amphibian Gastrulation
7.6 Cell movements during frog gastrulation
22
Amphibian Gastrulation
7.6 Cell movements during frog gastrulation
23
Amphibian Gastrulation
7.6 Cell movements during frog
gastrulation
7.7 Surface view of an early dorsal
blastopore lip of Xenopus
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Involution at the Blastopore Lip
• Dorsal lip of blastopore
– Margin cells turn inward and travel inside
animal hemisphere
– Form the lip
– Will become cells that involute to form
prechordal plate
• Precursor of the head mesoderm
– Followed by next cells to involute in at lip to
become chordamesoderm
• Will form the notochord
– Important to inducing and patterning nervous
system
25
Amphibian Gastrulation
7.8 Early movements of Xenopus gastrulation
26
Involution at the Blastopore Lip
– Slowly blastocoel is displaced to opposite
side of the dorsal lip
– Lateral lips and ventral lips form on
“crescent” to make additional mesoderm and
endodermal cells
• Finally a ventral lip covers over additional
mesodermal and endodermal precursor cells
• Forming a “ring” blastopore
– Remaining endoderm patch is called the
yolk plug
• Eventually internalizes
27
Amphibian Gastrulation
7.9 Epiboly of the ectoderm
28
Convergent Extension of the Dorsal
Mesoderm
• Convergent Extension of the Dorsal
Mesoderm
– Involution begins dorsally
– Pharyngeal endoderm and head mesoderm
– Next tissue to enter forms the notochord and
somite
– Lip of the blastopore expands to have
dorsolateraly, lateral, and ventral sides
• Prospective heart, kidney, and ventral mesoderm
that enters the embryo
29
Convergent Extension of the Dorsal
Mesoderm
– Blastopore lip IMZ cells (involuting marginal
zone)
• Several layers deep IMZ cells intercalate radially
• Continues to extend vegetally
• As these cells reach blastopore lip – involute
inwardly initiation 2nd intercalation
• Causes convergent extension along mediolateral
axis
– Simultaneous elongation with involution
– Mesoderm continues to migrate to animal
pole
• Forms an endodermal roof of the archenteron
30
Convergent
Extension of the
Dorsal
Mesoderm
7.10 Xenopus gastrulation
continues
31
Convergent Extension of the Dorsal
Mesoderm
– Central Dorsal mesoderm
• Become notochord and somites
– Remainder of body mesoderm
• Forms heart, kidneys, bones, …..
• Entered through ventral and lateral blastopore lips
• These create the mesodermal mantle
– Endoderm formed from involuting marginal zone
(IMZ) that forms lining of archenteron roof
– Endoderm from vegetal cells that become
archenteron floor
– Where endoderm meets ectoderm becomes anus
32
Epiboly of the prospective ectoderm
• Epiboly of the
prospective ectoderm
– The animal cal and
noninvoluting marginal
zone (IMZ) cells expand
by epiboly
• Covers the entire embryo
• Form the surface
ectoderm
• By increasing the cell
number (through
division) coupled with
concurrent integration of
several deep layers into
one
– Involves assembly of
fibronectin into fibrils
33
Epiboly of the prospective ectoderm
7.13 Epiboly of the ectoderm is accomplished by cell division and
intercalation
34
Progressive Determination of the
Amphibian Axes
• Remember
– This process whereby the central nervous
system forms through interactions with the
underlying mesoderm
• Called Primary Embryonic Induction
– A principle way vertebrate body becomes
organized
– Discoverers called the dorsal blastopore lip
• “The Organizer”
35
Hans Spemann and Hilde Mangold:
Primary Embryonic Induction
• Hans Spemann
– Began his most famous work in 1903
on demonstrating nuclear equivalence
– Later he won the Nobel Prize in 1935
– Later his work (1924) centered on the
dorsal lip cells, referred to as the
Spemann’s Organizer
• Hilde Mangold
– PhD Student of Spemann
– Began assisting him in his work in
1924 and contributed to understanding
cell fate in early gastrulation
– These cells originated as selfdifferentiating tissue of the dorsal lip
– Derived from the gray crescent
cytoplasm
36
Hans Spemann and Hilde Mangold:
Primary Embryonic Induction
•
Spemann Organizer
–
Role of the dorsal lip cells destined to be:
•
•
•
Dorsal mesoderm
Notochord
Some anterior pharyngeal
1. Induce host’s ventral tissue to change fate
and form neural tube and dorsal mesoderm
(such as somites)
2. Organize host and donor tissues into 2nd
embryo with AP and DV axes using donor
tissue
37
Hans Spemann and Hilde Mangold:
Primary Embryonic Induction
7.17 Organization of a
secondary axis by dorsal
blastopore lip tissue
38
Hans Spemann and Hilde Mangold:
Primary Embryonic Induction
• Spemann Organizer (cont)
– Responsible for neural tube formation
• Chordamesoderm & ectoderm cannot organize
the entire embryo
– Responsible for transforming flanking
mesoderm into AP axes
– These events initiate a sequential series of
sequential inductive events
– This is Key Inductive Event
• Dorsal lip cells inducing the A-P axis and neural
tube
• Called Primary Embryonic Induction
39
Molecular Mechanisms of
Amphibians Axis Formation
This actually created more questions than
answers by these experiments
• How does the organizer form?
– ~ dozen cells of the initial organizer
position themselves opposite the point of
sperm entry
– Now discovered that they are in the right
position for 2 signals converging
• First signal – tells the cells that they are dorsal
• Second signal – tells them they are mesoderm
40
The Dorsal Signal: -Catenin
Two signals & others:
1. Dorsal signal: -caterin
–
–
–
Isolating cap, marginal, vegetal cells
Organizer cells are special because they reside
above a special group of vegetal cells
Recombined animal cap with vegetal cells
•
•
•
•
–
Vegetal cells induced mesoderm formation from animal
cap cells (marginal/equatorial cells)
Ventral vegetal – blood, mesenchyme
Intermediate vegetal – muscle, kidney
Dorsal vegetal – somites, notochord
These dorsal inducing vegetal cells called the
Nieuwkoop center
41
The Dorsal Signal: -Catenin
7.18 Summary of experiments by Nieuwkoop and by Nakamura and
Takasaki, showing mesodermal induction by vegetal endoderm
42
The Dorsal Signal: -Catenin
β-caterin
Activin-like TGF-β &FGFs
7.18 Summary of experiments by Nieuwkoop and by Nakamura and
Takasaki, showing mesodermal induction by vegetal endoderm
43
The Dorsal Signal: -Catenin
– Proof of dorsal vegetal cells is
inducer of organization
• Nieuwkoop center
– Similar proof of dorsal-most
vegetal cells induce animal cell
to form dorsal mesoderm
– -caterin
• Protein acting as cell anchor for
cadherins
• Found to give this property to
dorsal vegetal cells
• [In sea urchin responsible for
specifying micromeres]
44
The Dorsal Signal: -Catenin
• If -catenin is originally found throughout
the embryo, how does it become localized
specifically to the side opposite the sperm
entry?
– Answer  translocation of Wnt11 and the
Disheveled (Dsh) protein from the vegetal pole
to the dorsal side of the egg at fertilization
– -catenin is targeted for destruction by glycogen
synthase kinase 3 (GSK3)
• GSK3 destroys b-catenin and blocks axis formation
• GSK3 is inactivated by GSK3-binding protein (GBP)
– Occurs during the 1st cell cycle when microtubules are
formed
45
The Dorsal Signal: -Catenin
7.21 Model of the mechanism by which the Disheveled protein
stabilizes -catenin in the dorsal portion of the amphibian egg
46
The Dorsal Signal: -Catenin
• GSK3-binding protein (GBP)
– Travels along the microtubules by binding to kinesin
– Moves to a point opposite sperm entry
– Disheveled grabs the GPB and is translocated on
microtubules
– Cortical rotation is probably in orienting and
straightening the microtubular array
– GBP and Dsh are released at the opposite point of
sperm entry
• Future Dorsal side of embryo
• They inactivate GSK3  allowing -catenin accumulation
• Ventral side GSK3 is degraded
47
The Dorsal Signal: -Catenin
7.20 The role of Wnt pathway proteins in dorsal-ventral axis
specification, -catenin (orange)
48
Function of the Organizer
• Nieuwkoop center cells remain endodermal,
cells of the organizer become dorsal mesoderm
and migrate underneath dorsal ectoderm
– Dorsal mesoderm induces central nervous system
formation
• Properties of organizer – 4 major functions
1. Ability to self differentiate dorsal mesoderm
2. Ability to dorsalize surrounding mesoderm into
paraxial mesoderm (somites)
3. Ability to dorsalize ectoderm forming neural tube
4. Ability to initiate the movement of gastrulation
49
Function of the Organizer
•
Organizer cells ultimately contribute to 4 cell
types:
1.
2.
3.
4.
•
•
•
Pharyngeal endoderm
Head mesoderm (prechordal plate)
Dorsal mesoderm (primarily notochord)
Dorsal blastopore lip
1 & 2 – induce forebrain and midbrain
3 – induces hindbrain and trunk
4 – remains to end of gastrulation then
becomes chordaneural hinge that induces the
tip of the tail
50
Induction of Neural Ectoderm and
Dorsal Mesoderm: BMP Inhibitors
• Problem:
– There is NO molecule secreted by the
organizer and received by the ectoderm
– How is neural tissue made from ectoderm?
• Organizer – ectoderm – neural tissue
– NO!
– It is the epidermis induced to form and
NOT the neural tissue
– Ectoderm is induced to become epidermal
tissue by binding to
• Bone Morphogenetic Proteins (BMPs)
51
Induction of Neural Ectoderm and
Dorsal Mesoderm: BMP Inhibitors
•
Nervous system forms from region of
ectoderm that is protected from epidermal
induction (BMP)
– By BMP-inhibiting molecules
1. The “default fate” of ectoderm is to become neural
tissue
2. Certain parts of embryo induce the ectoderm to
become epidermal tissue by secreting BMPs
3. The organizer tissue acts by secreting molecules
that block BMPs – thus allowing ectoderm
“protected” by BMP inhibitors to become neural
tissue
52
Regional Specificity of Neural
Induction
• Regional differences
– Regional specificity of neural structures
• Forebrain
• Hindbrain
• Spinocaudal region
– Organizers not only form neural tissue but
also specify regions
– Can be induced
53
Regional Specificity of Neural
Induction
Figure 7.29 Regional
and temporal
specificity of induction
54
Specify Left Right Axis
• Most parts symmetrical
• Heart and internal organs
are not
• Xenopus gene Xnr1
– Xenopus nodal related 1
• Expression of nodal gene in
the lateral plate mesoderm on
the left side of embryo
• Determine heart and gut side
• Clockwise rotation of cilia
keeps it to the left side
• Pitx2 activated by Xnr1
– Normally expressed on left
side and controls which side
heart and gut folds occur
7.36 Pitx2 determines the
direction of heart looping
and gut coiling
55