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Axis Specification and Patterning III
Axis specification in the frog
embryo (Xenopus laevis)
Amphibians were the first models for
studying vertebrate development
Studying these organisms has led to
formulation of “key” questions and
also provided some tentative answers
to these questions allowing vertebrate
developmental biology to flourish
Developmental history of the leopard frog
Fertilization
Egg accumulating yolk in vegetal
cytoplasm (stained green)
Fertilization is external and takes place
as the eggs are released by the female
A newly laid clutch of eggs with
distinct animal and vegetal poles
Prior to fertilization the frog egg is already polarized, the dense yolk is at the
bottom (vegetal pole) and the upper half has very little yolk (animal pole)
Certain mRNAs and proteins are already localized in specific regions of the
unfertilized egg
 Fertilization can occur anywhere in the animal
hemisphere
 The point of sperm entry determines dorsalventral polarity
 The point of sperm entry marks the belly
(ventral) side of the embryo
 The point 180° opposite to the point of sperm
entry marks the dorsal (spinal) side
 The sperm centriole organizes the microtubules
into parallel tracks in the vegetal cytoplasm
 The cortical cytoplasm now is separated from
the yolky central cytoplasm and rotates30° with
respect to it
 This exposes the grey cytoplasm (grey crescent)
opposite the sperm entry point. Gastrulation
begins here.
Unequal radial holoblastic clevage
First clevage
Second clevage
Fourth clevage
early blastula
late blastula
Cross section through
late blastula
16-64 cell stage is called a morula (mulberry like) and at 128 cell stage
the blastocoel becomes apparent
The blastocoel serves two functions:

It can change its shape to facilitate cell
migration during gastrulation.

It prevents premature interaction between cell
below with the cells above.
Mid-blastula transition preparing for gastrulation
• The major transcription of zygotic genes begins such that different genes
are transcribed in different cells
• The cell cycle acquires gap phases
• Blastomeres acquire the capacity to become motile
Goals of amphibian gastrulation
 To bring inside the embryo those areas
destined to form the endodermal organs.
 To surround the embryo with cells capable
of forming the ectoderm.
 To place the mesodermal cells in the
proper positions between the ectoderm
and endoderm.
Fate map at mid-blastula stage
Early movements of Xenopus gastrulation
 Vegetal rotation and invagination of the bottle cells: Internal cell rearrangements propel
the cells of dorsal floor of blastocoel towards animal cap (vegetal rotation). This places the prospective
pharangeal endoderm cells adjacent to the blastocoel and above the involuting mesoderm. In the
region of the grey crescent cells invaginate to form a slit-like blastopore. The bottle cells change their
shape dramatically.
 Involution at the blastopore lip: After the bottle cells have brought the involuting marginal
zone (IMZ) into contact with the blastocoel wall the IMZ cells involute into the embryo.
Amphibian gastrulation
Amphibian gastrulation
 As new cells enter the embryo the blastocoel is displaced to the side opposite the dorsal lip
 Meanwhile the lip expands laterally and ventrally as bottle cell formation and involution continue around the
blastopore
 The widening blastopore crescent develops lateral lips and finally a ventral lip
 With the formation of a ventral lip a ring has formed around the large endodermal cells called the yolk plug
The work of Hans Spemann and Hilde Mangold
Autonomous specification versus inductive interactions
Spemann’s demonstration of nuclear equivalence in newt cleavage
Spemann demonstrated that early amphibian nuclei are genetically identical
and capable of forming the entire embryo
Asymmetry in the amphibian egg
If a similar experiment was performed
with the constriction still longitudinal but
perpendicular to the plane of the first
cleavage (separating the future dorsal and
ventral halves rather than the left and
right sides) the result was very different.
The nuclei continued to divide on both
sides of the constriction but only one side
(future dorsal side containing the grey
crescent) gave rise to a normal larva. The
other side formed a mass of unorganized
tissue which Spemann named “the belly
piece”
Something in the region of the grey crescent was essential for proper embryonic development
The grey crescent region was essential for embryonic development
How?
Fate map showed that grey crescent region
gives rise to the dorsal blastopore lip where
gastrulation is initiated.
Spemann speculated:
 The importance of the grey crescent region lies in its ability to initiate gastrulation.
 Critical changes in cell potency occur during gastrulation.
Determination of ectoderm during newt gastrulation
 The ectoderm gives rise to the epidermis on one side
and neural ectoderm on the other side
 Replacement of presumptive epidermis of a nonpigmented strain with the presumptive neural
ectoderm of a pigmented strain produced a
pigmented epidermis when this was done in early
gastrula
 Replacement of presumptive epidermis of a nonpigmented strain with the presumptive neural
ectoderm of a pigmented strain produced a
pigmented neural plate when this was done in late
gastrula
Conclusion: In the early gastrula the transplanted tissue undergoes conditional development because the
ultimate fate depends on the location in the embryo. In the late gastrula the transplanted tissue exhibits
autonomous development independent of their final location after transplantation, i.e. their fate was
already determined.
Primary embryonic induction
The most spectacular transplantation experiment done by
Hans Spemann and his doctoral student Hilde Mangold
 Through transplantation of tissues from different regions
in the early gastrula they showed that the only region
which is autonomously determined is the dorsal lip of
the blastopore.
 Transplantation of the dorsal blastopore lip from a
pigmented newt early gastrula to the region that would
be the presumptive ventral epidermis “belly skin” in an
unpigmented gastrula showed that this tissue not only
continued to be the dorsal blastopore lip it could initiate
gastrulation and embryogenesis in the surrounding
tissue.
 The pigmented donor tissue continued to form the
chordamesoderm (notochord) and other mesodermal
tissue that it normally forms and also induced the
changes in the surrounding host cells to make tissues
that they normally would not have made.
Spemann’s organizer: Dorsal blastopore lip cells and their
derivatives the notochord and head endomesoderm
Called an “organizer” because:
 They induce the host’s ventral tissues to change their fates to form a
neural tube and dorsal mesoderm
 They organize the host and donor tissues into a secondary embryo
with clear anterior-posterior and dorsal-ventral axes.
However Spemann’s work together with the work of others has shown that this interaction of the
chordamesoderm and the ectoderm is not sufficient to organize the entire embryo.
Rather it initiates a series of sequential inductive events. This key induction in which the progeny of
the dorsal lip induce the dorsal axis and the neural tube is known as the primary embryonic induction.
Questions raised after Spemann’s organizer was identified
???
 How did the organizer get its properties?
 What makes the dorsal blastopore lip different from the rest of
the embryo?
 What factors secreted from the organizer cause the formation
of the neural tube and specification of the anterior-posterior
and dorsal-ventral axes?
 How did the different parts of the neural tube along the A-P
axis get established?
Molecular mechanism of amphibian axis formation
How does the organizer form?
Isolated animal cap cells become ectoderm, isolated
marginal/equatorial cells become mesoderm and
isolated vegetal cells become gut-like tissue
If animal cap cells are put in direct contact with
vegetal cells they become mesoderm. Thus vegetal
cells secrete factors that induce the mesoderm.
Experiments done by Pieter Niewkoop and by Osamu Nakamura and Takasaki
Nieuwkoop center: The dorsal most vegetal cells of the
blastula capable of inducing the organizer
Gimlich and
Gerhart in 1984,
transplanted the
dorsalmost
vegetal
blastomere from
one blastula to
into the ventral
vegetal side of
the other blastula
two embryonic
axes formed
Dale and Slack in
1987 recombined
single vegetal
blastomeres with
uppermost
animal tier of a
fluorescently
labelled blastula
of same stage,
the dorsalmost
vegetal cell
induced animal
pole to become
dorsal mesoderm
Transplantation and recombination experiments on Xenopus embryos demonstrate that the vegetal cells
underlying the prospective dorsal blastopore lip region are responsible for initiating gastrulation
What gives the dorsal most vegetal cells their special properties?
The Wnt pathway is involved and a major candidate is β-catenin
WHY?
Two types of experiments support the presence of a Wnt activity in the Nieuwkoop center:
1. Vegetal explants need the maternal Wnt pathway to induce expression of organizer and dorsal mesoderm
genes in recombined animal caps or equatorial explants.
2. Components of the Wnt pathway induce a secondary axis, or rescue the axis of UV-ventralized embryos,
when injected in vegetal cells that do not themselves form dorsal mesoderm, and thus behave like a
Nieuwkoop center
β-catenin is found throughout the embryo
as it is translated from the maternal
mRNA but it preferentially accumulates in
the presumptive dorsal side. In the
vegetal balstomeres the presumptive
dorsal side shows nuclear localization
whereas the ventral side does not.
Model of the mechanism by which the Disheveled protein stabilizes -catenin in the dorsal
portion of the amphibian egg
 Three proteins, Wnt11, GSK-3 binding protein (GBP) and
Dishevelled (Dsh) are translocated from the vegetal side
of the egg to the future dorsal side of the embryo during
fertilization.
 -catenin is targeted for destruction by GSK-3. In fact if
GSK-3 activity in ventral cells is blocked a secondary axis
forms.
 GSK-3 can be inactivated by GBP and Dsh. These two
proteins release GSK-3 from the degradation complex and
prevent it from binding -catenin and targeting it for
destruction.
 GBP binds to Kinesin a motor that moves towards the
growing end of microtubules i.e. away from the site of
sperm entry. Dsh binds to GBP and is thus transported
along with it.
 Thus in the future dorsal side of the embryo GBP and Dsh
inactivate GSK-3 and stabilize -catenin
However mere translocation of GBP and Dsh is not sufficient to
stabilize -catenin. Wnt activation is required. Wnt activity is provided
by Wnt 11. The mRNA for Wnt 11 is also localized to the vegetal cortex
and is transported to the future dorsal side by cortical rotation
Summary of events hypothesized to bring about
induction of the organizer in the dorsal mesoderm
 Microtubules allow the translocation of Dsh and Wnt
11 proteins to the future dorsal side of the embryo.
 Dsh binds GSK-3 thereby allowing -catenin to
accumulate in the future dorsal portion of the embryo.
 -catenin enters the nucleus and together with Tcf-3
activates the transcription of siamosis and twin.
 Siamosis and Twin interact with Smad2 transcription
factor activated by vegetal TGF-β family members
(nodal related proteins, Vg1, activin etc.)
 Together these transcription factors activate
“organizer” specific genes such as goosecoid, chordin,
noggin etc.
Specification of the germ layers
The vegetal cells have two major functions:
1. To differentiate into endoderm
2. To induce the cells immediately above
them to become mesoderm
 The mechanism of this “bottom up” specification resides in a set of mRNAs tethered to the vegetal cortex.
 The maternal mRNA for the transcription factor VegT is critical for both endoderm and mesoderm specification.
 The VegT mRNA is translated shortly after fertilization and in the vegetal blastomeres turns on the Sox17 transcription
factor required to specify endoderm fate.
 VegT also induces the expression of Nodal paracrine factors in the vegetal cells which act on the cells above them to
activate Smad2 by phosphorylation in these cells which then induce the expression of eomesodermin and Brachyury
which impart a mesodermal fate to the cells lying above the vegetal cells.
 The Eomesodermin and pSmad2 co-operate to transcribe zygotic VegT thus setting up a positive feedback loop needed
to maintain mesodermal fate.
 Another maternal mRNA for Vg1 localized to the dorsal region of the vegetal pole is translated. Vg1 is a Nodal-like
protein which induces dorsal mesodermal fate to the cells above it.
The vegetal induction of mesoderm
 The other factor that is critical for
activating the genes that
characterize the organizer cells is
phosphorylated Smad2 required
for mesoderm formation.
 Smad2 is activated in the
mesodermal cells when it
becomes phosphorylated
downstream of the Nodal related
paracrine factors secreted by the
vegetal cells.
 Activated Smad2 functions as a
transcription factor with a
partner.
 In the late blastula there is a gradient of Nodal- related proteins form dorsal (high) to ventral
(low) side. Vg1 which is also a Nodal-related protein acts in the same way by activating
The maternal RNA encoding
Smad2, hence it has a additive effect with the other nodal-related proteins.
Vg1 is tethered to the vegetal
 The dorsal high to ventral low gradient of nodal related proteins is largely produced by βcortex of a Xenopus Oocyte
catenin. In the future dorsal region β-catenin co-operates with Veg-T to increase the
transcription of Nodal-related genes.
 The dorsal high to ventral low gradient of Nodalrelated proteins is largely produced by β-catenin. In
the future dorsal region β catenin co-operates with
Veg-T to increase the transcription of Nodal-related
genes (Xnr1, 5 and 6).
 The more ventral blastomeres in the endoderm lack
the expression of these Nodal-related genes.
 In the region that will become the most anterior
portion of the organizer-pharangeal endodermhigher levels of Nodal-related proteins produces
highest activity of pSmad2. These induces the
transcription of hhex gene. Hhex together with Twin
and Siamosis (induced by β-catenin) specify
pharangeal endoderm to be foregut and to induce
anterior brain development in the ectoderm above.
 Slightly lower levels of pSmad2 activate goosecoid expression in cells that will become prechordal mesoderm and
notochord.
 Even lower levels of pSmad2 result in the formation of ventral and lateral mesoderm.
 Mesodermal regions with little or no Xnr have high levels of BMP4 and XWnt8.
In summary, the intersection between the vegetal pathway activating the Nodal-related
genes and pSmad2 in the mesodermal cells above them with the β-catenin activated
Twin and Siamosis on the future dorsal side leads to the “organizer” being specified.
The Nieuwkoop center is in the endodermal cells.
The “organizer” is in the mesoderm comprising of four different
tissues: pharangeal endoderm, head mesoderm (prechordal plate),
dorsal mesoderm (notochord) and the dorsal blastopore lip.
The properties of the “organizer”
 The ability to self differentiate into dorsal mesoderm ( prechordal plate, chordamesoderm etc.)
 The ability to dorsalize the surrounding mesoderm into paraxial (somite forming) mesoderm when it would have
differentiated into ventral mesoderm.
 The ability to dorsalize the ectoderm and induce formation of the neural tube.
 The ability to induce the movement of gastrulation.
What are the factors produced by the organizer?
Evidence showing that the organizer produces soluble factors which impart neural fate to the ectoderm
 If notochord fails to migrate beneath the
ectoderm it will not become neural tissue
rather it will become epidermis.
 Transfilter studies where neural structures
were induced in presumptive newt ectoderm
by newt dorsal lip tissue separated from
ectoderm by nucelopore filter with a pore
diameter of 0.05 mm.
Induction of the neural ectoderm and dorsal mesoderm: BMP inhibitors
Neural induction ?
Scientists were looking for molecules secreted
from the organizer that induce neural fate in
the ectoderm.
Wrong idea! As it turns out the default fate for
the ectoderm is to become neural tissue. BMPs
induces the ectoderm to become epidermis.
The organizer secretes BMP antagonists which
block this and allow a certain portion of the
ectoderm to acquire its default fate.
Three of the major BMP
inhibitors secreted by the
“organizer” are Noggin,
Chordin and Follistatin
The soluble protein Noggin dorsalizes the amphibian embryo
 In 1992 Smith and Harland constructed
a cDNA library from dorsalized (Lithium
chloride treated) gastrulae.
 Messenger RNAs synthesizied from sets
of these cDNAa were injected into
ventralized (UV-irradiated) embryos.
 Further splitting of those cDNA sets
that were able to dorsalize these
embryos into single cDNAs led to the
identification of Noggin.
Dosage-dependent rescue of
ventralized embryos by
injection of noggin mRNA
Expression of noggin mRNA:
i) Accumulted in dorsal marginal zone
ii) In the dorsal blastopore lip
iii) During convergent extension in the
precursors of pharyngeal endoderm,
prechoral plate and notochord.
iv) Extending beneath the ectoderm.
Noggin does two things:
1) It induces dorsal ectoderm to form neural
tissue.
2) It dorsalizes mesodermal cells that would
otherwise form ventral and lateral
mesoderm.
Localization of chordin mRNA
 Sasai et al. 1994, isolated
Chordin protein from clones of
cDNA whose RNA were present
in dorsalized but not ventralized
embryos.
 Confirmed the function of
Chordin by injecting
morpholinos targeting chordin
message which blocked
secondary axis formation by the
organizer graft.
Follistatin
 Follistatin is also transcribed in the dorsal blastopore lip and the notochord
 Follistatin was found in a screen unexpectedly looking for something else
 Ali Hemmanti-Brivanlou and Doug Melton wanted to see if Activin (a TGFβ
family member) was required for mesoderm induction
 In searching for the mesoderm inducer they found Follistatin which inhibits
both BMPs and Activin.
 Based on their studies they proposed the “default model” of neural
induction from ectoderm.
 Of all the organizer genes
chordin is the one most acutely
activated by β-catenin.
Epidermal inducers: The BMPs
BMP4 elicilts
expression of
different genes
in a
concentration
dependent
fashion
 In Xenopus the major epidermal inducers are the
bone morphogenetic proteins (BMPs) mostly BMP4
but also BMP2 and BMP7.
 There is an antagonistic relationship between
BMPs and the organizer.
 Initially BMP4 is expressed throughout the
ectoderm and mesoderm.
 During gastrulation Goosecoid protein and other
transcription factors induced by Siamosis, Twin and
pSmad2 in the dorsal region repress thanscription
of bmp4 and wnt8 expression in the dorsal region.
 In the ectoderm BMPs repress the genes (such as
neurogenin) involved in neural tissue formation.
 In the mesoderm graded levels of BMP4 activate
different mesodermal genes:
i) Absence of BMP4 specifies dorsal mesoderm
ii) A low amount specifies intermediate mesoderm
iii) High amount specifies lateral mesoderm
Control of neural specification by levels of BMPs
 Khokha and colleagues in 2005 used antisense
morpholinos to eliminate three BMP antagoists
Chordin, Noggin and Follistatin.
 The resulting embryos had catastrophic failure of
dorsal development,lacked neural plate and dorsal
mesoderm.
 Reversdale and colleagues blocked BMP2, 4 and &
simultaneously with antisense morpholinos the neural
tube became greatly expanded covering a much larger
region of the ectoderm.
 When they inactivated ADM ( another protein of BMP
family) along with BMP2, 4 and 7 the entire ectoderm
became neural
Regional specificity of neural induction
 The forebrain, hindbrain and
spinocaudal regions must be properly
organized along the A-P axis
 The organizer tissue not only induces
the neural tube it also specifies the
regional identity with the neural tube
 This was demonstrated by Otto
mangold in 1933, by transplanting fur
successive region of the archenteron
roof from the late gastrula of newt to
the blastocoel of early gastrula.
 Different anterio-posterior structures
were induced e.g. balancers and oral
apparatus, head structures nose, eye
etc, hindbrain and finally the dorsal
trunk and tail mesoderm
Temporal specificity of induction
 Mangold also demonstrated that
transplantation of dorsal lip from
early gastrula to blastocoel of
another early gastrula led to
formation of secondary heads
 Whereas transplantation of dorsal
lip from a late gastrula to and early
gastrula led to formation of
secondary tails.
What paracrine factors from the organizer provide this specificity?
Paracrine factor antagonists from the
organizer are able to block specific paracrine
factors to distinguish head from tail
Inhibiting Wnt signaling enables head formation
 Injecting Cerberus mRNA
into ventral D4 blastomere
induces head structures as
well as a duplicate heart and
liver. Xwnt8 is capable of
ventralizing the mesoderm
and preventing head
formation in the ectoderm
 Frzb is secreted version of
the Wnt receptor Frizzled
and hence can sequester
Wnt ligands and prevent
them from acting
 mRNA in situ for Frzb (dark)
and Chordin (red) showing
Frzb expression in the head
endomesoderm but not in
the notochord
Signaling gradients and axis specification
 A wnt signaling pathway posteriorizes
the neural tube. (A) shows gradient of βcatenin activity in the presumptive
neural plate.
 The Wnt gradient provides A-P polarity
and BMP gradient provides D-V polarity.
 FGFs seem to be critical for allowing
cells to respond to Wnt
 RA is also a major posteriorizing signal
Model of organizer function and axis specification in the Xenopus gastrula