Chapter 7

Xenopus Development
 Oogenesis

Follicle cells
Germinal vesicle
Oogonia in the frog’s ovary
persist until adulthood and
continue to divide
throughout life.
 GROWTH PHASE OF OOGONIUM
1. Accumulate ribosomes and transfer RNA
- Gene cluster coding for ribosomal RNA becomes amplified at an early
stage

2. Aquire a large amount of yolk proteins

- made by the liver of the female frog -> absorbed by oocytes from the
blood stream
- Preyolky oocyte ( transparent) becomes opaque due to yolk granules

3. Established animal-vegetal polarity

 GROWTH PHASE OF OOGONIUM
animal-vegetal polarity
2. mRNAs becomes associated with the Balbiani body
vegT
- becomes localized to the vegetal cortex
- Controls the localization of other mRNAs
- Antisense oligonucleotide treatment (degradation) caused other mRNAs to
become delocalized
3. Accumulation of pigment granules at the animal pole
hemisphere
vg1
-also becomes localized to the vegetal cortex

vegT

This transcription factor induces the expression of endoderm specific genes

(e.g. bix1-4, mix1-2, sox17)
Ectoderm is expanded in VegT depleted embryos, demonstrating
that VegT represses ectodermal fates.

Indeed, it is possible that ectoderm is a default state that forms

in the absence of maternal VegT.
 MATURATION PHASE OF OOGONIUM

- In response to gonadotrophins secreted by the pituitary gland

of the mother
- Travel to the blood stream and provoke release of
progesterone from ovarian follicle cells
 MATURATION PHASE OF OOGONIUM

1. Release of progesterone from ovarian follicle cells

2. Bind to steroid receptor in oocytes
3. Activates translation of c-Mos
4. C-Mos activates phosphatase Cdc25
5. Cdc25 activates M phase promoting factor /maturation
promoting factor (MPF)
- complex of Cdk and cyclin required to initiate M phase
6. Germinal vesicle breaks down , chromosomes condense, spindle
fibers formed
7. First meiotic division takes place
8. Gives rise to secondary oocyte and first polar body
9. Secondary oocyte arrested in metaphase II ( complex of c-mos
and Cdk2 ( cytostatic factor)- inhibits cyclin breakdown)
10. Eggs are shed into the body cavity
11. Enter the oviducts , travel down and become wrapped in jelly.
Maturation-promoting factor (abbreviated MPF, also called
mitosis-promoting factor or M-Phase-promoting factor) is the
cyclin-Cdk complex that was discovered first in frog.
It stimulates the mitotic and meiotic phases of the cell cycle.

MPF promotes the entrance into mitosis (the M phase) from

the G2 phase by phosphorylating multiple proteins needed
during mitosis. MPF is activated at the end of G2 by a
phosphatase, which removes an inhibitory phosphate group
added earlier.

The MPF is also called the M phase kinase because of its ability
to phosphorylate target proteins at a specific point in the cell
cycle and thus control their ability to function.
 FERTILIZATION- ZYGOTE

1. Secondary oocytes are fertilized by the sperm as the eggs

emerge from the cloaca (Zygote)

2. Rise in intracellular calcium

a. brings about the destruction of the cytostatic factor
(complex of c-mos and Cdk2) -> breakdown of cyclin->
b. progression into second meiotic division-> release of second
polar body
c. cause exocytosis of cortical granules -> lift the vitelline
membrane off the egg surface
 DEVELOPMENT- CLEAVAGE STAGE

• Fertilization is followed by cleavage, a period of

rapid cell division without growth

• Cleavage partitions the cytoplasm of one large

cell into many smaller cells called blastomeres

• The blastula is a ball of cells with a fluid-filled

cavity called a blastocoel
 DEVELOPMENT- CLEAVAGE

1. Sperm enters the animal hemisphere and initiates a cytoplasmic rearrangement –

CORTICAL ROTATION
- Appearance of oriented
array of microtubules in
the vegetal hemisphere

- Leads to a reduction in
the pigmentation of the
animal hemisphere on the
prospective dorsal side,
opposite the sperm entry
point

- Dorsal determinant is
moved from the vegetal
pole to the dorsal side
CORTICAL ROTATION

Good candidates for this dorsal determinant are proteins called

dishevelled (Dsh) and GSK3 binding protein (GBP). Both proteins
associate with the vegetal array of microtubules and become enriched
on the dorsal side of the embryo following cortical rotation.
 DEVELOPMENT- CLEAVAGE
Dorsal
Right

Anterior Posterior

Left
Ventral
(a) The three axes of the fully developed embryo

Animal pole Pigmented First

Animal Point of cleavage
cortex
hemisphere sperm
nucleus
entry Future
dorsal
side
Vegetal Gray
hemisphere crescent
Vegetal pole

(b) Establishing the axes

 DEVELOPMENT- CLEAVAGE

MID-BLASTULA TRANSITION (MBT)

The time at which significant transcription of the zygotic
genome commence

Early Domains of Zygotic genes

One of the earliest indicators of dorsal development is the accumulation of

beta-catenin in dorsal nuclei followed by activation of Wnt target genes,
such as Siamois, in the Spemann organizer.
 DEVELOPMENT- CLEAVAGE

T-box Brachyury – expressed in entire mesoderm

- needed to upregulate later mesodermal genes
- to control gastrulation movements

Siamois, goosecoid, not, lim1 – transcription factors

involved in the organizer region ( dorsal sector)
- required for axial differentiation
DORSAL
Siamois- involved in the formation of the organizer ( MESODERM
Spemann Organizer)
Goosecoid- involved in gastrulation

VENTRAL
Vent1 and vent 2 – homeobox genes MESODERM

Mix1 and sox7 ENDODERM

In 1924, the Ph.D. student Hilde Mangold working in the
laboratory of German embryologist Hans Spemann performed
an experiment that demonstrated that:

the pattern of development of cells is

influenced by the activities of other cells

(1869 - 1941)
A German embryologist who worked extensively on
amphibian development and was the discoverer of
the organiser region (or primitive node) the
controller of gastrulation.

Received the 1935 Nobel Prize in Physiology or

Medicine "for his discovery of the organizer effect in
embryonic development"
She cut out a piece of tissue from the gray crescent region of one newt
gastrula and transplanted it into the ventral side of a second newt
gastrula.

The remarkable results:

the transplanted tissue developed into a second
notochord
neural folds developed above the extra notochord
these went on to form a second central nervous system
(portions of brain and spinal cord) and eventually
a two-headed tadpole.
 The Spemann organizer
- The dorsal blastoporal lip region of
an amphibian embryo that emits BMP
inhibitors and thereby dorsalizes the
surrounding tissue
- blocks the action of BMP-4 by
secreting molecules of the
proteins chordin and noggin
- The organizer diffuses dorsalizing factors
that counteract the effects of the
ventralizing BMPs.
Niewkoop center
- the dorsal- and vegetal-most cell of the early blastula
- Region of the early amphibian embryo that induces
Spemann organizer via the Wnt pathway
 Cells on the ventral side of the blastula secrete
a variety of proteins such as bone
morphogenetic protein-4 (BMP-4) -
induce the ectoderm above to become
epidermis.

The Spemann organizer blocks the action

If their action is blocked, the
ectodermal cells are allowed to follow the surface of the overlying ectoderm cells.
their default pathway, which is to
become nerve tissue of the brain and
spinal cord.
of BMP-4 by secreting molecules of the
proteins
chordin and Noggin
Both of these physically bind to BMP-4
molecules in the extracellular space and thus
prevent BMP-4 from binding to receptors on

In the Spemann/Mangold experiment, transplanting an organizer to the

ventral side provided a second source of chordin. This blocked BMP-4
binding to the overlying ectoderm and thus changed the fate of those cells to
forming a second central nervous system rather than skin.
 DEVELOPMENT- GASTRULATION
- Phase of morphogenic movement that brings about the
formation of the three germ layers
• The three layers produced by gastrulation are called embryonic
germ layers
– The ectoderm forms the outer layer
– The endoderm lines the digestive tract
– The mesoderm partly fills the space between the endoderm and
ectoderm

• Gastrulation in the frog

– The frog blastula is many cell layers thick
– Cells of the dorsal lip originate in the gray crescent and
invaginate to create the archenteron
– Cells continue to move from the embryo surface into the
embryo by involution
– These cells become the endoderm and mesoderm
 DEVELOPMENT- GASTRULATION
INVAGINATION – infolding of a cell sheet to form an internal protrusion or pocket
INVOLUTION- infolding of a cell sheet my movement led by a free edge

- Phase of morphogenic movement that brings about the

formation of the three germ layers
To some extent independent of each other

1. EPIBOLY – active expansion of the animal hemisphere

2. INVAGINATION of the marginal zone – starts on the dorsal side and

spreads to the lateral and ventral sides until the blastopore is circular.

• Archenteron- cavity formed

• Yolk plug becomes internalized at the end of gastrulation. – becomes
part of the archenteron floor.
 DEVELOPMENT- GASTRULATION

3. As invagination proceeds, the mesoderm separates from the endoderm and

INVOLUTES as a separate tissue layer between the ectoderm and endoderm.

4. ELONGATION of the dorsal axial mesoderm in the anteroposterior direction.

By convergent extension – cells becoming polar

- actively moving in between their neighbors
- elongation at right angles to the direction of cell
movement
- involves the small GTP proteins rho and rac
( activated by the Wnt planar polarity pathway – by wnt5a as dominant ligand)
 DEVELOPMENT- GASTRULATION

The Wnt/Frizzled pathway is a major pathway implicated in the

specification of cell and tissue polarity and operating in different developmental
processes, including heart and neural tissue development, kidney morphogenesis,
limb polarity and sex determination.

key role for Wnt signaling in early dorsoventral asymmetries and axis specification.

involved in neural induction and anteroposterior neural patterning later

in development.
A 'canonical' Wnt signaling pathway has been elucidated in vertebrate
and invertebrate model systems. In the canonical pathway, Wnt binding leads
to the stabilization of the transcription factor β-catenin, which enters the
nucleus to regulate Wnt pathway target genes.

However, Wnt binding also acts through β-catenin-independent,

noncanonical pathways, such as the planar cell polarity (PCP) pathway and a
pathway involving Ca2+ signaling.
 planar cell polarity (PCP) pathway
These noncanonical pathways have also been termed the Wnt/Calcium and
Wnt/JNK pathways in vertebrates and the Wnt/planar cell polarity pathway (PCP)
in flies.
function in a β-catenin independent manner to regulate processes such as
convergent extension during vertebrate gastrulation

Parallel Rac and Rho pathways

downstream of Wnt/Fz/Dvl signaling.

Dsh is a cytoplasmic phosphoprotein

that acts directly downstream of
frizzled receptors.

Rho family of GTPases that

cycle between the inactive
GDP-bound form and the
active GTP-bound form and
regulate diverse cellular
processes such as
cytoskeletal dynamics, cell
adhesion,
Wnt/Fz signaling induces Rac and Rho activation during cell-cycle
Xenopus
gastrulation.
Cold Spring Harbor Laboratory Press
progression
 ECTODERM
Nervous tissues
Epidermis
Lens and ear
Sense organs

MESODERM
Head mesoderm
notochord
somites-> Muscles
Bone
Cartilage
Circulatory system ( blood,
blood vessel, heart)
Reproductive (gonads)
Excretory (kidney)

ENDODERM
Digestive organs ( epithelial
lining of the gut, liver, pancreas,
bladder)
At the end of gastrulation, the embryo has a Respiratory organs (lungs)
true ANTEROPOSTERIOR AXIS Endocrine glands
 DEVELOPMENT- GASTRULATION
If embryos are placed in a salt solution of osmolarity similar to blastocoel

Gastrulation is deranged.
Instead of invagination into the anterior, the endomesoderm
evaginates from the ectoderm. – EXOGASTRULATION

Ectoderm and endomesoderm is normal but the CNS is defective

and there is no tail
 DEVELOPMENT- NEURULATION

• Early in vertebrate organogenesis, the notochord forms from

mesoderm, and the neural plate forms from ectoderm
Genes expressed in the whole neural plate includes: sox2 and N-
CAM

• The neural plate soon curves inward, forming the neural tube
• The neural tube will become the central nervous system (brain
and spinal cord)

Neural crest cells develop along the neural tube of vertebrates and form
various parts of the embryo (nerves, parts of teeth, skull bones, and so on)

• Mesoderm lateral to the notochord forms blocks called somites

• Lateral to the somites, the mesoderm splits to form the coelom
 REGIONAL SPECIFICATION

Dorso-ventral patterning

Germ Layer Formation

Antero-posterior patterning

Head endoderm
 REGIONAL SPECIFICATION
DETERMINANTS of the fertilized egg:

1. Ventral determinant
- Cause the formation of the endoderm

Source of mesoderm-inducing signal

- Induce a ring of tissue around the equator
to become the mesoderm

2. Dorsal determinant
- Causes the formation of the organizer

- source of dorsalizing signals that induce the neural

plate and pattern the mesoderm into zones forming the tissue
- consists of components of the WNT signaling
pathway

3. Germ plasm
- appears in the Balbiani body of the stage II oocyte -
end up in the vegetal cortical region
- specify primordial germ cells , contain cat2 mRNA
 REGIONAL SPECIFICATION

Dorso-ventral patterning
- After fertilization, the dorsal determinant is moved from the
vegetal pole to the dorsal side by cortical rotation
- Depends on an array of microtubules
- Dorsal determinant consists of components of the canonical Wnt
signal transduction pathway – causes the stabilization of beta-
catenin
- Wnt5a and wnt11
* If wnt pathway components are overexpressed in early embryos->
results in a HYPERDORSAL embryo ( too much head and not enough
trunk and tail)
- Immediate target of β- catenin are the transcription factor Tcf1

Becomes activated as an activator from a

repressor by association with β- catenin.

Net result is the upregulation of dorsal sector organizer genes: Goosecoid

and bone morphogenic protein (BMP) inhibitors
 CANONICAL WNT SIGNALING PATHWAY
Wnts activate cell surface receptors belonging to the frizzled family, which
transduce the signal across the plasma membrane to Dsh.

Activated Dsh, and GBP, interact with a protein complex that includes the enzyme
glycogen synthase kinase 3 (GSK3),

which phosphorylates ßcatenin and targets it for degradation. Dsh and GBP
inactivate GSK3, allowing ßcatenin to accumulate.

Net result is the upregulation of dorsal sector organizer genes: Goosecoid

and bone morphogenic protein (BMP) inhibitors

ßcatenin accumulates in nuclei on the dorsal side of the embryo at blastula stages,
forming complexes with the transcription factor XTcf3,
and activates transcription of dorsally expressed genes such as siamois, twin, and xnr3.

siamois, twin two closely related homeodomain transcription factors that

induce secondary dorsal axes
The Spemann organizer
- The dorsal blastoporal lip region of an
amphibian embryo that emits BMP
inhibitors

dorsalizes the surrounding tissue

- blocks the action of BMP-4 by

secreting molecules of the proteins
chordin and noggin

- The organizer diffuses dorsalizing factors

that counteract the effects of the
ventralizing BMPs.

What will happen if ß-catenin mRNA is Injected into ventral

blastomeres? induces a second dorsal axis on the ventral
side of the embryo.
 REGIONAL SPECIFICATION
Germ Layer Formation
MESODERM

A key criteria for a native mesoderm inducing factor is that it

must be localized to the vegetal hemisphere of blastulae.
vg1
Inducing factor of the nodal class, and not a transcription factor

How to demonstrate if indeed vg1 has a role in

mesoderm induction?
- inject antisense oligonucleotides into oocytes
mesoderm induction is impaired in embryos
depleted of maternal vg1 mRNA

maternal signals are not sufficient for mesoderm induction in Xenopus and that
zygotic signals are also required
 REGIONAL SPECIFICATION
Germ Layer Formation
MESODERM

mesoderm inducing factor vegT

- necessary both for endoderm and mesoderm formation

VegT depleted vegetal poles have no mesoderm inducing activity when

grafted to animal poles, despite containing Vg1

transcription factor and not a secreted protein, it cannot be directly

responsible for activating mesodermal gene expression in the marginal
zone
controlled by maternal VegT and therefore localized to the vegetal
pole of blastulae:

TGF-ß family, such as derrière, xnr1, xnr2, xnr4, xnr5, and xnr6
(Xenopus has six nodal-related genes but xnr3 has no mesoderm
inducing activity. Mammals have only one nodal gene).
 REGIONAL SPECIFICATION

MESODERM

Transcripts for xnr1, xnr2, and

xnr4 are first detected in the
dorsal half of the vegetal
hemisphere and expression
remains higher than in the
ventral half throughout blastula
stages.

Thus high concentrations of

Xnrs could account for the
ability of dorsal-vegetal
blastomeres, the Nieuwkoop
centre, to induce dorsal
mesoderm
 MODELREGIONAL SPECIFICATION
FOR MESODERM INDUCTION

At the gastrula stage, the organizer secretes a cocktail of factors that

refine the initial patterning
dorsal-specific genes, such as goosecoid, in the dorsal marginal zone, while
low levels of Xnrs induce expression of ventrally expressed genes
such as xbra
activates zygotic expression of the TGF-ß family members derrière,
xnr1, xnr2, and xnr4 in the vegetal hemisphere
 REGIONAL SPECIFICATION
MESODERM
establish just two territories within the marginal zone

small dorsal sector (90˚ of arc) that differentiates dorsal-type mesoderm

(notochord)
larger ventrallateral sector that differentiates ventral-type mesoderm
(blood)
The remaining mesodermal tissues - muscle, pronephros, and lateral plate
- are specified by signals that are released from the dorsal mesoderm,
a process known as dorsalization.

The dorsal mesoderm also releases signals that induce the

overlying ectoderm to form neural tissue, a process known as
neural-induction.

Dorsalization and neural-induction were first demonstrated in

the experiments of Hans Spemann and Hilde Mangold (1924),
 REGIONAL SPECIFICATION
Germ Layer Formation
ENDODERM
- Determinant for endoderm : VegT

Induce the expression of endodermal transcrption factors:

sox17 , mix1 , gata4

- Endoderm becomes regionalized into zones that will form the

different tissue types of the gut and respiratory system

Upregulation of transcription factors: pdx1 cdx

Specification
an embryonic cell somehow gets
information that tells it how to
differentiate

Determination
cells have made a commitment
to a differentiation program.
VegT (also known as Antipodean [Stennard et al. 1996], Xombi [Lustig et al.
1996], Brat [Horb and Thomsen 1997], and XTbx6r [Smith 1997]), in early
development.

- a member of the T-box family of transcription factors, the founder-member of

which, the mouse T gene (Brachyury), is necessary for the development of the
notochord and somites posterior to somite seven ( Smith 1997; Papaioannou and
Silver 1998, for review).

Related proteins have been found in many animal groups and are known to play
roles in cell lineage specification. Maternal VegT mRNA is localized in Xenopus to
the vegetal hemisphere of the full-grown oocyte and is inherited by cells that
develop from this region of the egg ( Lustig et al. 1996; Stennard et al. 1996;
Zhang and King 1996).

Zygotic transcription of VegT begins before gastrulation and is restricted to

equatorial, presumptive mesodermal cells, particularly on the dorsal side.
Overexpression and ectopic expression studies show that VegT mRNA can induce
both mesoderm and endoderm, while a dominant negative form inhibits
mesoderm formation and severely disrupts normal development ( Horb and
Thomsen 1997).