Patterning the Vertebrate Body Plan I: Axes and Germ Layers
Wolpert L, Beddington R, Jessell T, Lawrence P, Meyerowitz E, Smith J. (2001) Principles of Development. 2th ed. London: Oxford university press.
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Principles of Development. 2th ed. London: Oxford university press.
Gilbert SF. (2003) Development Biology. 7th ed. Sunderland: Sinaure Associates Inc.
The vertebrate body plan
The vertebrate body plan consists of the antero-posterior axis (segmented vertebral column and skull) and the dorso-ventral axis (including the ventrally located mouth).
All vertebrate embryos pass through the phylotypic stage when the embryos are all similar in appearance.
These embryos share 1) the head,
2) the neural tube (under which is the notochord) and 3) mesodermal somites (flanking the notochord)
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3) mesodermal somites (flanking the notochord) .
The large eggs of fish, frogs and bird have large yolks that provide nutrients to the developing embryo.
Mammalian eggs are small and obtain nutrients from the ovoduct then the placenta.
Phylotypic stage
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The three main stages of vertebrate development (pattern formation)
1) setting up the main body axes (1.A/P; 2.D/V; 3.R/L, bilateral) 2) specification of three germ layers (endoderm, mesoderm and
ectoderm) ectoderm)
3) germ layer patterning (mesoderm and early nervous system)
Maternal genesprovide factor (RNA and proteins) to the egg during oogenesis (including sub-cellular localization to specific regions. It expressed in the mother during the development of
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regions. It expressed in the mother during the development of the egg.
Zygotic genesare expressed by the embryo's genes.
Both maternal and zygotic genes may have long term effects upon the embryo's development.
Xenopus animal/vegetal axis is maternally determined
Before fertilization (in mother), the animal pole being different from the vegetal pole is clearly set up.
Animal pole: pigmented (no function),uppermost, contain nucleus, Animal-model, amphibian
Vegetal pole: unpigmented,
Holoblastic, displaced radial cleavage (mesolecithal)
Cleavage of the embryo divides the contents of the egg into distinct regions.
1st cleavage - parallel to (with) the axis (Animal-vegetal) (vertical division) about sperm entry Nieukwoop center
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division) about sperm entry-Nieukwoop center
2nd cleavage - 90° to 1st cleavage (vertical division) 360/2 =180, 90 3rd cleavage - 90° to both above cleavages to separate the animal
from the vegetal poles (horizontal division). 23=8
Animal-vegetal axis of the egg is certainly related to the antero- posterior axis
Summary of the main patterns of cleavage
Lecithal
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The cleavage of Xenopus egg
The 3th division, only placed in animal pole
Fig 10.1-2
16-64 cell is called a morula; 128 cells formed blastocoel, also call blastula
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After the 3th division, the size discrepancy between the animal and
vegetal cells 4th division (16 cells)
Cell fate in Xenopus
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Isolated blastomere fate in Xenopus
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Axis formation in Xenopus
Animal vegetal axis
- originates during oogenesis
- will develop into the anterior posterior axis during gastrulation
Dorsoventral
- forms after fertilization and fixed before the first cleavage
Left-right symmetry
-not recognizable until early organogenesis will see as internal structures are laid our
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will see as internal structures are laid our
Maternal mRNAs and proteins include:
1) abundant housekeeping proteins which are concerned with 'normal' cellular activities.
2) small amounts of mRNAs or Protein with developmental roles Xenopus animal/vegetal axis is maternally determined
2) small amounts of mRNAs or Protein with developmental roles.
Box 3A Intercellular signal-protein
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Technique: In situ hybridization
Purpose: To determine when and where a particular gene's mRNA is present.
Details: Complementary single-stranded nucleic acid strands (RNA p y g ( or DNA), commonly known as "probes", will bind tightly (hybridize) to a specific mRNA.
This can be visualized if the probe is labeled (or tagged) with an isotope, a fluorescent dye or an enzyme that produces a coloured substance.
When embryos (or another tissue) are fixed permeabilized and
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When embryos (or another tissue) are fixed, permeabilized and then allowed to undergo such a hybridization the pattern of gene expression is revealed.
Whole mount and tissue sections can be visualized.
Technique: In situ hybridization
Principle Whole mount
DNA A=T
tissue sections A=T
C=G
−
mRNA A=U
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tissue sections A U
C=G
−
Xenopusanimal/vegetal axis: localized mRNAs Localized mRNAs control determination of the animal/vegetal axis
in Xenopus.
Many are signaling molecules which specify early polarity and induction of the mesoderm.
Vg-1 of the TGF-beta (transforming growth factor) family of growth factors.
Vg-1 is synthesized during early oogenesis and is localized to the vegetal cortexof the oocyte. → It is maternally signaling By early after fertilization, Vg-1 has moved to the vegetal
cytoplasm to drive animal/vegetal identity.
The animal-vegetal axis of the egg is certainly related to the antero- posterior axis of the tadpole ,
Fig. 3.2 Vg-1 distribution 14
As the head forms the animal region.
Effect of VegT mRNA depletion on germ layer development in Xenopus
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vegT→ mesoderm ↓
Dorso-ventral axis of amphibian is determined by the site of sperm entry
In Xenopus, before fertilization, the egg has a radial symmetry.
Sperm entry sets up the D/V axis with the dorsal side of the embryo at a place opposite of the sperm entry point.
Within the first 90 minutes after fertilization, the cortex rotates approximately 30 degrees and is toward the site of sperm entry approximately 30 degrees and is toward the site of sperm entry.
The cortex is a gel-like layer of actin filaments about 5 micrometres thick just under the membrane.
The vegetal cortex opposite the sperm entry point moves towards the animal pole.
This leads to the formation of a 'signaling centre' opposite the site of sperm entry known as the Nieuwkoop Centre.
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Cortical rotation in amphibian eggs
Gray crescent
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Cortical rotation moves membrane bound organelles and dorsalizing activity from the vegetal pole to the dorsal side.
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Cell movement during Xenopus gastrulation
archenteron
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Mesoderm induction in Xenopus blastula
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The importance of the Nieuwkoop Centre
Nieuwkoop Centre sets up D/V polarity in the blastula.
The 1st cleavage cuts through the site of sperm entry and the Nieuwkoop Centre.
Th 2 d l lit th b i t 4 ll (2 ith th Ni k
The 2nd cleavage splits the embryo into 4 cells (2 with the Nieuwkoop Centre and 2 without).
sperm
1st cleavage
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Nieuwkoop center
Experiment 1: Divide the 4 cell embryo into two halves, one dorsal and one ventral.
The half with the Nieuwkoop Centre develops fairly normally missing only some ventral structures.
The half without the Nieuwkoop Centre develops very abnormally to produce a radially symmetric embryo without dorsal or anterior structures
The importance of the Nieuwkoop Centre (Dorsal-ventral)
a radially symmetric embryo without dorsal or anterior structures.
No dorsal and anterior Fig.3.4 The Nieuwkoop centers is essential for normal development (Dorsal part)
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First cleavage, passes through sperm entry-Nieuwkoop center axis → Nieuwkoop left and right → bilateral symmetry
Second cleavage, divides the egg into the ventral and dorsal axis
No gut
Nieuwkoop Centre: dorsal and anterior structures
• Experiment 2: Nieuwkoop Centre grafts.
1) When cells of the Nieuwkoop Centre are grafted onto the ventral side, twinned embryoswith two 'dorsal sides’ are produced.
2) When cells of the ventral side are grafted onto the dorsal side, normal embryos are produced (no effect).
Therefore, signal from the Nieuwkoop Centremust be required for developing dorsal and anterior structures.
To recall Roux’s experiment, a hot needle was used to kill one of two embryonic cells (but it was not removed).
If the cell would have been removed (separated) then regulation would have
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(separated) then regulation would have lead to a small embryo.
Nieuwkoop center
Transplantation experiments on 64-cell amphibian embryos demonstrating that the vegetal cell underlying the prospective dorsal blastopore lip region are responsible for causing the initiation of gastrulation
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Disruption of the Nieuwkoop Centre
Blocking rotation (and Nieuwkoop Centre formation) by exposing the embryos to ultraviolet light (which disrupts microtubules) produces
"ventralized" embryos which have an excess of ventral blood-forming mesoderm (Nieuwkoop centre is important role of induction of dorsal mesoderm. (Nieuwkoop centre is important role of induction of dorsal region)
In general, an increase in U.V. light results in a decrease in dorsal and anterior structures.
The effects of U.V. can be rescued by:
1) re-orienting the eggs to mimic cortical rotation or
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2) grafting Nieuwkoop Centre cells from other embryos.
Dorsalized embryos can arise from treatment with lithium.
Both treatments probably interfere with proteins involved in formation of the D/V axis.
Cortical rotation and the Nieuwkoop Centre Cortical rotation specifies the Nieuwkoop Centre.
The signal from the sperm entry to form the Nieuwkoop Centre is unknown. It may be related with egg’s cytoskeleton However, formation of the Nieuwkoop Centre depends upon
rotation of the cortex.
Th t ti h t f th t l i h b
The rotation changes a part of the vegetal region, perhaps by more contact with the animal cytoplasm.
The factors help designate the embryo's midline. To establish the plane of bilateral symmetry of the future embryo.
The midline passes through the entry point and the Nieuwkoop Centre.
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Fig. 3.6 Cortical rotation toward the point of sperm entry
Nieuwkoop Centrespecifies the Spemann Organizer
The Nieuwkoop Centre's main role is to specify another signaling centre…
the Spemann Organizer which in turn leads to the development of A/P and D/V axes.
Sperm entry results in the D/V axis and involves an external signal.
Egg structure determines the A/P and then the D/V axes by internal signals.
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g
Near Neiuwkoop region (by dorsal blastopore) is plays an important role of Organization of axis
Nieuwkoop
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Nieuwkoop center →Vegetal signal → Dorsal side (away from sperm entry) formed Mesoderm formation is induced by vegetal region
sperm entry) → formed Spemann organizer
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Vegetal cell → release ventral signal (TGF-b, FGF)
→ margin cells →induced mesoderm formation (even animal region)
Where is the regional specificity of mesoderm induction ? Demonstrated by recombining blastomeres of 32-cells embryo. Animal
pole cells were labeled with fluorescent polymers. Animal pole + individual vegetal blastomeres.
Area 1 most important for dorsal part
D1 was the most likely to induce the animal pole cells to form dorsal 31
mesoderm p induction
In the 32-celled embryo, the dorsalizing activity is strongest in B1, then C1 and then D1
Meridian
and then D1
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Molecular mechanisms of dorsolventral axis formation and mesodermal induction.
What are the factors that segregate to the dorsal blastomeres?
What are the inducers that persuade marginal cells to form mesoderm What are the inducers that persuade marginal cells to form mesoderm rather than ectoderm?
- must be present in the embryo at the required concentration - must be present in the appropriate location
- must be maternally supplied if before MBT
-must be synthesized from embryo genome if after MBT
if bl k th i ti i i h ld bl k i d ti
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- if we block their action in vivo – should block induction - should be able to rescue embryos depleted of the activity by
adding the activity back.
MBT: mid-blastula transition
Beta-catenin
- leading candidate for the signal of the Nieuwkoop center
Catenins - proteins that anchor cadherins to the cytoskeleton Ca e s p o e s a a c o cad e s o e cy os e e o Cadherins - transmembrane proteins that function in cell adhesion
Beta-catenin – however
- is a cytoplasmic protein and is involved in the Wnt signaling pathway.
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Wnt - soluble ligand (paracrine factor) - binds to receptors of the frizzled family
Specific pathway (Wnt) in dorsal-ventral axis specification
Dorsal view ventral view
-catenin -catenin in nuclear
E: injection b-catenin in 2 cell → formation a new dorsal axis
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dorsal axis
-catenin dorsal localization persists through the gastrula stage
Ca2+ dependent homophilic cell-cell adhesion in adherens junctions and desmosomes is mediated by cadherins
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Protein constitutents of typical adherens junctions
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Cytosolic domains of the E-cadherin bind direct or indirectly to multiple adapter protein that connect the junctions to cytoskeleton and participate in intracellular signaling pathways (catenin)
The effect of Maternal protein on dorsalizing and ventralizing
Maternal proteins involved in D/V effects.
Maternal proteins can act as in the Nieuwkoop Centre or normal and U.V.
treated embryos.
beta-catenin (cell adhesion and Wnt signaling) Fig 3 7
signaling) Fig. 3.7
Accumulates on the dorsal side following cortical rotation, dorsal-high, ventral-low Injection of bata-catenin can induced new
dorsal region Fig. 3.8
Beta-catenin→prevention degrade → into nucleus →help transcription factor → turn no siamois gene → specific dorsal
Enter nucleus
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g p
development
Nieuwkoop center is to specify another key dorsal signaling center-Spemann organizer
Spemann organizer are involved the A/P,
D/V and central nervous system Fig. 3.7
Fig. 3.8
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The effect of Maternal protein on dorsalizing and ventralizing
Xwnt-11 and Vg-1 (partial Nieuwkoop Centre activity)
The formation of the Nieuwkoop Centre is caused by the suppression of ventralizing signal including GSK-3.
Lithium directly inhibits GSK-3. ß-catenin and other members of the Wnt signaling pathway act to inhibit GSK-3
signaling pathway act to inhibit GSK-3 Fig. 3.9
Wnt or Vg-1 → Dsh→ Axin→
prevent b-catenin degradation → turn on dorsal gene transcription
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GSK: glycogen synthase kinase TCF: transcription factor
Dorsal structure development
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Cortical rotation → translocation of Dsh → move to dorsal region → Dsh + GSK-3 → -catenin accumulate → catenin enter to nuclear and bind to Tcf-3 protein The hypothesis of induction of the organizer in the dorsal mesoderm
p formation a complex →
transcription factor→ activate gene encoding siamois protein→→
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Dorsal formation
Chick blastoderm: the axes depend on gravity
The blastoderm is a disc of cells which sits on top of the yolk.
The D/V axis depends upon the dorsal side forming away from the yolk while the ventral side is next to it.
I iti ll di ll t i ft th i l id d ll f t th
Initially, radially symmetric after the egg is laid, dense cells form at the posterior marginal zone (PMZ).
The primitive streak develops from the PMZ.
The egg rotates through the hen's uterus (in the oviduct) (once every 6 minutes) as the embryo develops. And cleavage has already started at this stage. Fig 3.10
The blastoderm (uppermost) contains thousands of cells when the egg is
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laid.
The posterior marginal zone can be though of as an organizing center (like Nieuwkoop center in Xenopus), it can induced new axis (Fig.
3.11)
Fig. 3.10
Egg rotation by gravity Direction: X → Y Axis is P → A
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Fig. 3.11 PMZ is posterior and induced new A/P axis
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posterior marginal zone (PMZ) = Nieuwkoop center
Xenopus
The different formation of the Nieuwkoop center in forgs and chicks
Chick
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Chick
-catenin is found in the outer cells of the blastobisc Vg-1 producing cells are restricted (probably by gravity) to the
presumptive posterior cells of the blastoderm.
Nieuwkoop
Chick blastoderm: primitive streak formation
As the egg rotates, the embryo develops depending upon the influence of gravity. A/P axis
The egg is directed point down, the PMZ is at the top. D/V axis The rotation causes the blastoderm to ‘tip’ in the direction of rotation
(somewhat like an ‘off-balanced’top).
The primitive streak acts like a Nieuwkoop Centre in that it can induce a new axis if transplanted to another embryo
.
Chick Vg-1 is expressed in the primitive streak.
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Cells that express Vg-1 can induce a new primitive streak
.
Mouse embryo axes: cell-cell interactions not from mother
Mouse embryos have no apparent early polarity and no well ordered pattern of cleavage. No evidence for localized maternal factors affecting later development. (differ chick and Xenopus)
The second polarity body and the point of sperm entry may define axes in The second polarity body and the point of sperm entry may define axes in
the fertilized that are related to future axes in the blastocyst. (chick and xenopus were before fertilized)
At the 32 cell blastocyst stage, the position of the cells determine their cell fate.
Cell-cell interaction plays an important role of the development of AXIS.
Fig. 3.12
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By labeling cells, it was determined that outside cells contribute to the trophectoderm and inside cells give rise to the inner cell mass (ICM) that give rise to the embryo.
Mouse embryo axes: cell-cell interactions
Fig. 3.12
The specification of the inner cell mass of mouse embryo depends on the relative position of the cell with respect to the inside and
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to the inside and outside of the embryo
ICM: inner cell mass
Different position → different development
Animal-vegetal axis in the zygote, polar body = animal pole
At 4 day, (blastocyst stage), the inner cell mass is confined to the embryonic, or dorsal, region and this defines as embryonic-abembryonic axis.
The D/V axis relates to the position of the ICM within the blastocyst.
The D/V axis runs from the embryonic pole, where the ICM attaches to the Mouse embryo axes: is regulative and establishment at early stage
e / a s u s o e e b yo c po e, e e e C a ac es o e trophectoderm, to the opposite end.
The D/V axis persists until gastrulation starts.
Embryonic pole : The blastocyst has one distinct axis running from the site where the inner cell mass is attached Embryonic-abembryonic axis = dorso- ventral axis it related to the site of
Asterisks is polar body
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ventral axis, it related to the site of sperm entry. The sperm entry site coming to lie on the boundary between the embryonic and abembryonic halves.
Bilateral symmetry The green cell is injected with a long
lasting marker RNA for trace their fate
animal pole
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4 ½ days after fertilization, inner cell mass → differentiated to primary and primitive endoderm (on blastocoelic surface)→ implant →trophectoderm proliferates → produced extra- embryonic ectoderm → pushes the embryonic ectoderm → pushes the ICM across the blastocoel → formed proamniotic cavity → formed within the epiblast (proliferate)→epiblast grow to embryonic ectoderm→formed U shape.
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Amnion
V-D
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Relationship between the animal-vegetal axis of the egg and the embryonic –adembryonic axis of the blastcyst.
V D
The formation of antero-posterior axis First sign of the A/P axis at 5 1/2day (before gastrulation) epiblast cells → form primitive streak →primitive streak occur in posterior toward to anterior and formed the organizer
Epiblast was from ectoderm
Anterior par induced by the extra-embryonic anterior visceral d d
endoderm
Ventral visceral endoderm (express gene Hox) move anteriorly
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Allantois Amnion Parietal
Organ handedness in vertebrates Vertebrates are bilaterally symmertic (eyes, ears….) Most internal organs are asymmetric in positioning.
The condition, situs inversus, found in 1 in 10,000 people, is a mirror reversal of the 'handedness' of the internal organs.
Left versus right depends upon the A/P and the D/V axes and is under Left versus right depends upon the A/P and the D/V axes and is under
genetic control.
In chick, several genes are expressed asymmetrically with respect to Hensen’s node.
Fig. 3.15 Handedness may
be the asymmertic distribution of
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some protein.
Organ handedness in vertebrates
In the mouse, the iv gene controls the handedness of internal organs.
In iv mutants, organ handedness is random and some individuals show heterotaxis where normal and inverted organs are found in one animal.
Heterotaxis : The organs of normal and inverted asymmerty are present
Normal mutant
Heterotaxis : The organs of normal and inverted asymmerty are present in the same animal
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Zebrafish: shield determined antero-posterior asix
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Germ layers specification
The specification of the germ layers, 1)Endoderm→ gut, internal organ
2)Mesoderm→give rise to notochord muscle heat kidney and blood 2)Mesoderm→give rise to notochord, muscle, heat, kidney, and blood 3)Ectoderm→ epidermis and nervous system
is the earliest patterning of the embryo with respect to the body axes.
Fate map, shows the tissues of each germ layer come form, but did not indicates that specified or determined (for development plasticity).
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Regulation can be extensive in vertebrate embryos.
Fate mapping techniques:
1) stain parts of embryo with lipophilic dye
2) injection of high MW (molecular weight) dyes (i.e. rhodamine dextran)
... then observe final location of dye. 62
It can predict the germ layer come form
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Fig. 3.17 Fate mapping of the early Xenopus embryo; C3, is labeled by injecting fluorescein-dextran-amine which fluoresces green under UV light.
Fate of early regions of the Xenopus embryo
In the Xenopus embryo ...
1) The vegetal region gives rise to endoderm.
2) The animal pole becomes ectoderm (epidermis, nervous tissue).
3) The marginal zone (equatorial region) becomes mesoderm (bone, muscles, notochord etc.). The signal from vegetal pole.
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In Xenopus, a thin layer of endoderm covers the mesoderm.
Gastrulation is essential to move interior structures inside.
Fate of early regions of the Xenopus embryo The marginal zone moves inside the embryo through the dorsal
blastopore (above the Nieuwkoop Centre).
Early regions become specific types of tissues.
dorsal mesoderm → notochord
b d l d it ( l )
sub-dorsal mesoderm → somite (muscles)
sub-ventral mesoderm →lateral plate (heart and kidney)
ventral mesoderm → blood islands (also form in more dorsal regions) ventral ectoderm → epidermis (covers embryo after neural tube forms) dorsal ectoderm → nervous system
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Fig. 3.18 Fate map of late Xenopus blastula
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Xenopus / Zebrafish embryo: specification map
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Fig. 3.19 Specification map of a Xenopus late blastula
Fig. 3.22 Fate map of zebrafish at the early gastrula stage
Chick embryo: specification map
In the chick, early blastoderm cannot be mapped.
As the early chicken embryo is small and there is much cell proliferation and extensive cell (differ Xenopus) and tissue movements, the early fates are not known., y
Later, when the primitive streak forms, the three germ layers can be mapped.
Like the amphibian embryo, three germ layers (ectoderm, endoderm and mesoderm) can be identified.
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Fig. 3.20 Fate map of a chick embryo when the primitive streak has fully formed. Almost all the endoderm has already moved through the streak to form a lower layer.
Primitive streak formed the mesoderm and endoderm
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mesoderm
somites Cell ingressed
Mouse embryo: specification map The embryo cannot be mapped until 4 to 4½
days.
The primitive endoderm, which forms extra- embryonic tissues, is mapped to the outer cells of the inner cell mass.
The primitive ectoderm (which forms the embryo) develops between the primitive endoderm and the polar trophectoderm.
The ability to regulate demonstrates the state of the cell’s determination.
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Fig. 3.21 Fate map of a mouse at the late gastrula stage
Fig. 3.23 The fate map of vertebrate embryo (dorsal view)
Note:
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Note:
Future notochrod mesoderm occupies a central dorsal position
The neural ectoderm lies adjacent to the notochord, with the rest of the ectoderm anterior to it.
Types of cell movement during gastrulation
Invagination Involution
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Involution Ingression Delamination
Eiboly: ectoderm covers embryo
Cells of early vertebrate embryo do not yet have their fates determined
Powers of regulation, parts of the embryo are removed or rearranged.
Some experiment, many cells are not yet determined or specified.
Patterning mechanism involving cell interaction Using this characterization: for gene transfer, or chimera
Chimera: are mosaics of cell with two different genetic constitution-by fusing two embryo
Cell-autonomous: If only the mutant cell exhibit the mutant phenotype and are not rescued by the normal cells, the gene is acting cell-autonomously;
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a gene product not influencing other cells. (only one mutant → affect phenotype; directly)
Non-cell autonomous: when either the mutant cells in the chimera appear to act normally or the normal cells start to show the mutant phenotype. It action typically due to a gene product that is secreted by on cell and acts on the other. (one mutant cell → affect other cell → affect phenotype)
Fig. 3.24
Fusion of mouse embryo gives rise to a chimera
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Chimeric mice (Transgenic mice) To studying the role of a interesting gene in
development
Two way: 1. injection DNA into the nucleus of fertilized egg; 2. ES cell injection produced chimeric animal
Injection of inner cell mass cells from one mouse blastocyst to another will contribute to many tissues or phenomena.
Injection of genetically modified embryonic stem cells (ES cells) into a mouse blastocyst allows formation of transgenic chimeras which may breed to produce heterozygous (or
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y p yg (
homozygous) transgenic mice strains.
Homologous recombination techniques can replace a gene with a defective (deleted) version of the gene to produce a “knock-out”
mutant mouse strain.
Induction of mesoderm in Xenopus from the vegetal region and endoderm, ectoderm is specified by maternal factors
Mesoderm: vegetal region induced; Endoderm and ectoderm is maternal determined
Early patterning involves both cell-cell interaction and localized maternal factors. Specified by maternal factor in the egg Animal pole explants form ectoderm.
Vegetal pole explants form endoderm.
Animal pole explants cultured in contact with vegetal tissue produces some mesodermal tissue.
Th f d d d i l f th t l i t t
p y gg
Specific maternal transcription factor VegT → endoderm development
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Therefore, mesoderm depends upon signals from the vegetal region to turn animals pole cells from ectodermal fate to a mesodermal fate.
If animal and vegetal tissues are separated by a filter, then mesoderm develops.
Fig. 3.25
In gastrulation stage, different region develop different germ layer and specific tissue
Animal contact vegetal → d l t d ti The equatorial cell (animal
and vegetal resions are adjacent) form mesodermal tissues
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mesoderm related tissue
Mesoderm induction in Xenopus by a diffusible signal
A diffusible signal must travel but not by cell-cell contact. (separated by filter, still induction mesoderm development)
Different ages of embryonic tissues do not induce a signal. (Time specific)
specific)
Tissues at specific times (only in gastrulation stage) are competent to receive signals. (Fig. 2.27, Timing effect)
Cells must be present in sufficient number (200, at least) to be induced and represents a community effect in the responding cells. (Fig. 2.26, small numbers of cell did not induce)
Several signals induce and pattern the Xenopus embryo (Fig 3 28)
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Several signals induce and pattern the Xenopus embryo. (Fig. 3.28) After induction of the mesoderm, dorsal marginal zone explants become
notochord and muscle and mimic gastrulation movements (as normal).
Ventral and lateral mesoderm become mesenchyme and blood-forming tissues but not muscle (these normally form a lot of muscle).
Fig. 2.27, Timing effect
An intrinsic timing mechanism controls the expression of mesoderm-specific genes
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Fig. 2.26, small number of cell did not induce
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Fig 3.28, Specification map vs. Fate map
Many
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Many
specific/different signal regulate
The four different signal involves the induction of mesoderm
Two signal from vegetal region, two signal is related with ventral mesoderm (D/V axis)
Class 1) General mesoderm inducer from the vegetal region makes
“broad” ventral mesoderm, is ground or default state.
Class 2) Dorsal mesoderm inducer from the vegetal region from the Class 2) Dorsal mesoderm inducer from the vegetal region from the
Nieuwkoop center, it generates Spemann Organizer and notochord.
Class 4) Ventral mesoderm patterning signal (from spemann organizer), modulates the ventralizing signal (class 4), and from the ventral region subdivides muscle,kidney and blood. It also able formed a new dorsal axis. (Fig. 3.31)
Class 3) Spemann Organizer signal,and from dorsal region modifies the patterning of ventral mesoderm. Interacts with the dorsalizing signal.
82 Fig. 3.29
Late blastula stage
Class 4
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Beta-catenin
- leading candidate for the signal of the Nieuwkoop center
Catenins - proteins that anchor cadherins to the cytoskeleton Cadherins - transmembrane proteins that function in cell adhesion
Beta-catenin – however
- is a cytoplasmic protein and is involved in the Wnt signaling pathway.
Wnt - soluble ligand (paracrine factor)
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Wnt soluble ligand (paracrine factor)
- binds to receptors of the frizzled family
Wnt signaling pathway
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Cortical rotation → translocation of Dsh → move to dorsal region → Dsh + GSK-3 → -catenin accumulate catenin enter to The hypothesis of induction of the organizer in the dorsal mesoderm
accumulate → catenin enter to nuclear and bind to Tcf-3 protein formation a complex →
transcription factor→ activate gene encoding siamois protein→→
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Dorsal formation
-catenin mRNA injection induces a new dorsal side
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- during cortical rotation, associated with the microtubules that move the cortical components
Summary of evidence for b-catenin being responsible for Nieuwkoop center activity?
- UV irradiation blocks cortical rotation and traps b-catenin in vegetal pole - also, no activation of Siamois+ and twin+
- in the blastoderm stage, -catenin is enriched in the nuclei of dorsal but not ventral blastomeres
- removal of b-catenin from eggs blocks dorsoventral axis formation - inject antisense oligonucleotides against b-catenin mRNA
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inject antisense oligonucleotides against b catenin mRNA - ventralized embryo (via UV irradiation) can be rescued by microinjection of -catenin mRNA
Vegetal region signal regulated the induction of mesoderm
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Dorsal vegetal region contain Nieuwkoop center → notochord, muscle from animal cup
Ventral vegetal region contain → blood, associate tissue from animal cup, somites
Different vegetal region → induced different mesoderm related tissue (may be different signal from vegetal region)
Fig.3.31 Class 3 signal, transplantion of the spemann organizer induces a new axis
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Maternal signal expression: Mid-blastual transition in Xenopus Xenopuseggs are stuffed with maternal mRNA and proteins At fertilization, protein synthesis increase by 150% from the stored
maternal mRNAs. However, little new m-RNA synthesis. All protein are translation from maternal mRNA.
Zygotic gene expression begins at the mid blastula stage Zygotic gene expression begins at the mid-blastula stage.
Cell division loses its synchrony. Cycle time ↑, cell number slow increase
Possibly based on DNA quantity at this time.
Suppression of cleavage, but not DNA
th i i thi t h th
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synthesis in this stage, perhaps the titration of an unknown
transcriptional repressor leads to zygotic transcription.
How is the mid-gastrulation triggered? Timing mechanism
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Suppression of cleavage, but not DNA synthesis in this stage
Cell cleavage → cytoplasm X, DNA ↑ → some repressor in cytoplasm did not enough → RNA synthesis ↑
Candidate mesoderm inducers in Xenopus
1) Vg-1 (TGF-ß family) precursor must be proteolytically processed.
Mature and active Vg-1 can induce dorsal mesoderm in a) isolated animal caps and
b) ventralized (UV treated) embryos (ventral ) b) ventralized (UV-treated) embryos (ventral )
At lower concentrations it induces ventral mesoderm and may therefore be both the Class 1 and Class 2 signal.
2) activin (also TGF-ß family) may also be a candidate a mutant form (dominant negative mutant) of the activin receptor
blocks mesoderm formation. (Fig. 3.36)
Different concentration of maternal signal regulated development
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Vg-1 High →dorsal mesoderm Low →ventral mesoderm
Activin High notochord Low muscleAnimal cup
Fig.3.34 Signal transduction pathway of TGF- family
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Fig. 3.35 A different (gradient ) in the nodal-related protein may provide the class one and two signals in mesoderm induction
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VegT, Vg-1, catenin and nodal related protein are important in the induction of
mesoderm Activin?? 96
Fig. 3.36
A mutant activin receptors blocks mesoderm induction
m-RNA→ protein → function
Dominant-negative mutation
↓
un-correct m-RNA injection
↓
un-correct protein
↓
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↓ no function
Mesoderm patterning factors in Xenopus
A number of protein involved in patterning the mesoderm along the dorso- ventral axis. Fig 3.37; 38
BMP-4 (also TGF-ß family) induces ventral mesoderm (Class 4 signal) and is expressed throughout the late blastula.
Xwnt-8 (another diffusible factor) induces ventral mesoderm (Class 4 signal) Xwnt-8 (another diffusible factor) induces ventral mesoderm (Class 4 signal)
and is expressed in the future mesoderm.
Fibroblast growth factor (FGF) also induces mesoderm.
noggin can dorsalize mesoderm tissue but can not induce mesoderm from ectoderm (Class 3 signal).
frizbee can dorsalize mesoderm (Class 4 signal).
noggin (and chordin) bind BMP-4 and frizbee binds Xwnt-8 which prevents
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noggin (and chordin) bind BMP 4 and frizbee binds Xwnt 8 which prevents these factors from binding to their receptors.
Fig. 3.38
different signal involves the induction of mesoderm
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Fig. 3.39 Expression of noggin in the Xenopus blastula
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Mesoderm induction activates genes that pattern the mesoderm
Mesoderm induction activates zygotic mesoderm patterning genes, control mesoderm differentiation.
Brachyury, a transcription factor, is expressed throughout the mesoderm then becomes confined to the notochord (Fig 3 40) mesoderm then becomes confined to the notochord. (Fig. 3.40) Brachyury → FGF family expression
Overexpression of Brachyury in the ectoderm induces ventral mesoderm. High dose Brachyury induced the formation of muscle
Goosecoid, a transcription factor, is expressed in the Spemann organizer (dorsal mesoderm) and can largely mimic the Spemann transplantations
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Spemann transplantations.
Other transcription factors that are expressed in the Organizer region include:
Pintallavis, HNF-3 (two forkhead proteins), Xnot and Xlim-1 (homeodomain proteins)
a gene expressed in the organizer and midline cells of frog embryos: Involvement in the development of the neural axis
Fig. 3.40 Fig. 3.41
Zygotic gene expression specification map
A ti th h th
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A cross-section through the embryo along the animal-vegetal axis shows that Brachyury (red) is expressed in the future mesoderm
Zygotic gene expression in a late Xenpous blastula.
Several transcription factors are expressed in the region of the dorsal mesoderm that corresponds to the spemann organizer
Gradients in signaling proteins and threshold response could pattern the mesoderm
Different concentration induce different/specific protein
Different [activin] → animal cap cell Medium [activin] → animal cap → induced
Brachyury expression
High [activin]→ animal cap → induced
Fig 3.42 103
goosecoid expression
Low [activin] → animal cap →induced epidermis
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105 106
107
Mesoderm is induced, why?
different signal involves the induction of mesoderm
Nieuwkoop center vs. spamnan organizer (maternal factor vs. zogotic factos) abembryonic-embryonic axis
How identify maternal gene or zygotic gene How identify maternal gene or zygotic gene Blastocyte vs. embryonic stem cell in mammal Important of mid-gastrula stage
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