CHAPTER 7: XENOPUS

INTRODUCTION

- African Clawed Frog

- Scientific name: Xenopus laevis
- In vitro fertilization is very common
- Induced spawning: Injection of both male and female with chorionic gonadotrophin
- Diameter of early embryo: 1.4mm
- High yolk content: small multicellular explants survive and differentiate for several days in very
simple media
- Fluorescent dextrans: used for cell lineage studies and accurate fate maps
- Overexpression of genes are carried out by making synthetic mRNA in vitro and injecting it into
the fertilized egg.
- Morpholinos are used to inhibit gene action.
- Transgenesis enabled Xenopus to be used for molecular genetic studies of organogenesis,
regeneration, and metamorphosis.
- How xenopus gene and protein names should be written: prefixed with X and x. This
indicates their species of origin.

I. OOGENESIS, MATURATION AND FERTILIZATION

Frog ovary:
- Consists of oocytes surrounded by layers of follicle cells and blood vessels
- Oogonia continue to divide and become Primary Oocytes after their last mitotic division
*Growth of oocyte takes several months
- Oocyte nucleus
-> AKA Germinal vesicle
-> very large
- Chromosomes
-> AKA Lamp-brush chromosomes
-> four-stranded bivalents characteristic of meiotic prophase
-> remain transcriptionally active during oocyte growth
-> display numerous protruding loops of chromatin

Protein synthesis: ribosomes and transfer RNA are accumulated

-> Gene cluster coding for ribosomal RNA becomes amplified (about 1000 additional
extrachromosomal copies, all active as templates for rRNA synthesis)
->Large amount of yolk proteins is acquired by the liver of the mother and absorbed by the
oocytes from the blood stream.

Previtellogenic (preyolky) oocyte begins as transparent and becomes opaque due to yolk granules
*When the oocyte is fully grown, transcription ceases but protein synthesis and degradation
continues.
 Animal-vegetal polarity arises
-> Mitochondrial cloud:
- AKA Balbiani Body
- Precursor of germ plasm: its location determines the future vegetal pole of the egg.

The following mRNAs become localized to the vegetal cortex:

Veg T
Controls localization of other mRNAs
antisense oligonucleotide treatment cause other mRNAs to become delocalized
Vg 1
second group that appears when there is pigmentation difference

Animal Hemisphere: contains the germinal vesicle

- becomes dark due to an accumulation of pigment granules, while the vegetal pole
remains light.

Fully grown diameter of oocyte: 1.2 mm

MATURATION PROCESS
Achieved in response to gonadotrophins

1.Gonadotrophins provoke release of progesterone from ovarian follicles.

2.Bind to steroid receptors in the oocyte
3.Activate translation of oncoprotein cMos
4.cMos activates cdc25
5.Cdc25 activates maturation promoting factor (MPF)/ M-phase promoting factor
Complex of Cdk and cyclin required to initiate M phase
6.Breakdown of germinal vesicle
7.First meiotic division takes place
8.Formation of secondary oocyte and first polar body
9.Secondary oocyte arrested in metaphase II (due to the inhibition of cyclin breakdown by c-Mos with
Cdk2)
10.Eggs are shed into the body cavity, enter the oviducts, travel down and become wrapped in jelly.

FERTILIZATION:ZYGOTE
Male clasps female and fertilizes egg as they emerge from the cloaca.
Secondary oocyte becomes fertilized eggs (zygote)
Rise in intracellular calcium
1. brings destruction of cytostatic factor -> breakdown of cyclin -> progression into second
meiotic division -> release of second polar body
2. Cause exocytosis of cortical granules near egg surface -> allows egg to rotate freely under
gravitation and bring animal hemisphere uppermost.
 NORMAL DEVELOPMENT

Normal Development
Developmental substages:
Cleavage
Gastrulation
Neurulation
*Complete development of Xenopus: 24 hours at 24 degrees Celsius
> Nieuwkoop and Faber: devised a numerical stage series
Stage 8: Mid-blastula
Stage 10: Late gastrula
Stage 13: Early Neurula
Stage 24: End of neurulation
Cortical Rotation: cytoplasmic rearrangement initiated by sperm entry
- rotation of the egg cortex relative to the interior
- associated with the transient appearance of an oriented array of microtubules in the vegetal
hemispheres
- leads to the REDUCTION IN THE PIGMENTATION OF THE ANIMAL HEMISPHERE
(dorsal side, opposite to the sperm entry point)
Dorsal Determinant: moved from the vegetal pole to the dorsal side
- ensures that the dorsal structures will develop opposite the point of sperm entry
Grey Crescent: similar pigmentation in other amphibian species; surface feature

A. CLEAVAGE
First Cleavage: vertical; separates left and right hemispheres
Second Cleavage: vertical (right angle to the first cleavage); separating dorsal from ventral halves
Third Cleavage: equatorial; separating animal to vegetal hemispheres
Blastomeres: large cells resulting from early cleavage divisions
Blastocoel: cavity forms in the center of the animal hemisphere
Blastula: resulting embryo form cleavage division
>Outer surface of the blastula: consists of original oocyte plasma membrane
>Complete network of tight junctions: around the exterior cell margins that seals the blastocoel from the
exterior and renders the penetration of almost all substances highly inefficient.
>Oocyte: permeable up to stage of maturation to substances (amino acids and sugars)
>Internal cells of blastula: connected by cadherins and are readily dissociated by removal of calcium ions
from the medium
>Desmosomes: are not found until neurula stages
Gap Junctions: connects all the cells of early cleavage stages
Mid-Blastula Transition/MBT: cleavage continues rapidly for 12 hours
- actually occurs at the late blastula stage
- RATE OF CLEAVAGE SLOWS DOWN, SYNCHRONY OF CELL DIVISION IS LOST, and
INCREASE IN THE STRENGTH OF INTERCELLULAR ADHESION
- significant transcription of zygotic genome commences: possible detection of low level
transcription of some genes during the cleavage stage.
> T-box gene brachyury: visualization of entire mesoderm which is needed to upregulate later
mesodermal genes and to control gastrulation movements.
Organizer Region/Spemanns Organizer: in the dorsal sector that is characterized by the expression of
a variety of transcription factor genes (siamois, goosecoid, not & lim1)
siamois: involved in the original formation of the organizer
goosecoid: needed for gastrulation movements
vent1 and vent2: homeobox genes that expresses by ventral mesoderm that occurs in nested
pattern
vent1+2: specify lateral plate
vent2: specify somatic regions
mix1 and sox17: visualization of endoderm that are later required for upregulation of various
genes of particular endoderm derived tissues.

B. GASTRULATION
- phase of morphogenetic movements
Marginal Zone: belt of tissue that will become internalized through the blastopore.
>Start of Gastrulation: marked by the appearance of a pigmented depression in the dorsal vegetal
quadrant.
>Blastopore: becomes elongated laterally and soon becomes a complete circle
>Dorsal Lip: dorsal segment
>Ventral Lip: ventral part
>Bottle cells: elongated cells that are present around the blastopore
Several components of gastrulation movements
1. Epiboly: active expansion of animal hemisphere that eventually covers the whole embryo surface
2. Invagination: movement of the marginal zone. Starts on the dorsal -> spreads to the lateral and
ventral side -> blastopore is circular
Archenteron: cavity formed by invagination and expands at the expense of the blastocoel
3. Involution: mesoderm separates from endoderm and ectoderm
4. Convergent Extension: elongation of dorsal axial mesoderm that is an active process of
intercalation which helps drive the internalization of the marginal zone and the closure of the
blastocoel
5. Movement of the ventrolaterla mesoderm: towards the dorsal midline
>Rho and rac: small GTP exchange proteins that is required in convergent extension
- dominant (-) versions: block the process
- activated by Wnt planar polarity pathway with wnt5a: dominant ligand
>Paraxial protocadherin: cadherin-type adhesion molecule that is expressed in the paraxial mesoderm and
helps to generate activation of rho and rac through its cytoplasmic domain.
>Wnt Ca pathway: regulates cell adhesion during gastrulation, with the dorsal cells undergoing
convergent extension -> INCREASE INTRACELLULAR CALCIUM
Anteriorposterior axis: runs in the leading edge of mesoderm (anterior) to the residual blastopore
(posterior).
Exogastrulation: endomesoderm evaginates from ectoderm; dumbbell-like structure
- Ectoderm and endomesoderm: normally patterned
- Central Nervous System: substantially defective
 - NO TAIL

C. NEURULATION AND LATER STAGES

Neurula: ectoderm on the dorsal side becomes the central nervous system
Neural plate: becomes visible as a keyhole shaped region covering much of the dorsal surface of embryo
and delimited by raised neural folds.
sox2: associated with neural stem cells and neural cell adhesion molecule
Neural tube: arises from neural folds that moves together which after closure, becomes covered by
ectoderm from beyond the folds (epidermis)
>Striking elongation of the body: whole trunk and tail region are derived from the posterior quarter of the
neurula
- driven by a continuing process of convergent extension both in the notochord and other tissues
Axis: neural tube, notochord and somites
Tailbud stage: all major body parts are in their final positions
Notochord: forms from the dorsal midline of the mesoderm
Somites: appear on either side
>Lateral plate mesoderm: gives rise to limb buds, kidney and coelomic mesothelium
> String of blood islands: ventroposterior region of the mesoderm that provides erythrocytes
Neuroenteric canal: connection between neural tube lumen and gut
Proctodeum: terminating in the anus that forms from the channel between neuroenteric canal and exterior
formed by the process of neural tube closure
Three vesicles formed in the anterior neural tube:
forebrain
midbrain
hindbrain
Placodes: ectodermal thickenings
Nasal placode: consisting of sensory cells from which originate the axons of the olfactory nerve
connecting to the telencephalon
Lens placode: forming the lenses of the eye
Otic placode: form the ears
Optic lobes: outgrowths of the forebrain that develop eyes
Pigmented epithelium: forms from the outer layer
Retina: forms from the inner layer
Optic tecta: projection in which the optic nerve grows back to the retina down the optic stalk
Neural Crest Cells: migratory tissue that forms a variety of tissue types
- head: forms the skeletal tissues of the skull
- trunk: form the dorsal root ganglia and sympathetic gangia and melanocytes
- vagal and sacral regions of the trunk: forms the parasympathetic regions of the gut
>Head mesoderm: forms part of the jaw muscles and branchial arches
>Cement gland: develops from the anterior epidermis
>Heart: formed from the ventral edges of anterior lateral mesoderm

D. FATE MAPS
- published for amphibian embryos at stages form the fertilized egg to the end of gastrulation
Vital staining: neutral red or Nile blue which applied to embryo surface from a small fragment of
impregnated agar
Lineage labels: injection of horseradish peroxidase or fluorescein-dextran-amine which can fill the whole
cells and not diffuse
DiI: for surface marking that do not become diluted and so remain clearly visible for several days

Important features of fate map:

1. Neural plate: arises from the dorsal half of the animal hemisphere and the epidermis from the
ventral half
2. Mesoderm: arises from a broad belt around the equator of the blastula much from the animal
hemisphere
3. Endoderm: arises from vegetal hemisphere
4. Somitic muscle: arises from most of the marginal zone circumference, much from the ventral half
of the blastula
5. Dorsal and ventral structures at the anterior end of the body: come from the dorsal side of the
blastula

II. EXPERIMENTAL METHODS

Attempts to establish the function of any gene product

Expression pattern
In situ hybridization
Biological activity
Effect of specific inhibition in vivo
Injection of materials
Microsurgical isolation of explants
Ultraviolet irradiation

Gain of Function

1. Overexpression of specific RNA

Injection of synthetic mRNA

For biological activity measurement
mRNA to be injected is prepared in vitro
From plasmids with promoters for RNA polymerases of bacteriophage
Sp6, T3, T7, poly(A) addition site
Injected into a whole fertilized egg or into a specific blastomere
Remains active throughout early development

In situ hybridization
 For expression studies
Detection of a specific RNA, or occasionally DNA, in a biological specimen by
hybridizing to a specific probe with a suitable detection method
Probe: a labeled and antisense nucleic acid that can be used to detect the
complementary nucleic acid by hybridization
The same plasmid can be used for preparation of the in situ probes required by
transcription from te oppositely oriented promoter

Induction of gene activity at a particular stage

Protein of interest
added with the hormone-binding domain from the glucocorticoid receptor
binds to the cytoplasmic protein hsp90 sequestered in the cytoplasm
a glucocorticoid, usually dexamethasone, is added
Lipid-soluble
Can penetrate embryo
glucocorticoid binds to the receptor
protein is liberated from the hsp90
protein moves to the nucleus
transcription factor of the molecule exerts its biological activity

Transgenesis
Late development
Injected RNA may have been degraded
Plasmid DNA is incorporated into swollen sperm heads
Injected into unfertilized eggs
DNA is injected into fertilized eggs in the presence of a transposase or a rare-
cutting restriction endonuclease
A GFP coding sequence is incorporated into the transgene
To identify the transgenic embryos by their green fluorescence
Each individual transgenic embryo will have a different insertion site and copy
number
Most founders are used as founders
Causes some variability of biological behavior compared with a pure-
bred transgenic line

Effects of overexpression of a specific RNA are often not very informative due to the
nonspecific nature of the defects observed

Combined overexpression with another technique:

2. Autoinduction
Embryo is injected with a component of the mesoderm- or neural-inducing systems
Animal cap is explanted
Animal cap: animal pole region of the blastula
 Explanted animal cap will autonomously undergo induction
Some or all of the cells in the cap make and secrete the factor
All the cells are competent to respond to it
Untreated animal caps
spherical balls of epidermis
Induced to form axial tissues
undergo convergent extension
become very elongated
Induced to form ventral-type tissues
swell
form translucent vesicles
Observed under the dissecting microscope
Simple and convenient

3. Animal cap assay

For the study of extracellular factors
Proteins are applied to explants
Must be applied to the explant before it rounds up and becomes re-sealed
Outer surface of the embryo is impermeable
Can be through mRNA injection or treatment with a protein
Visual scoring of the results can be supplemented by conventional histology or
biochemical methods for specific mRNAs
In situ hybridization
Immunostaining for specific markers

4. Ultraviolet rescue
It is possible to create embryos with no axial structures by UV radiation
Injection of mRNA can restore a supposedly lacking part of an embryo which was treated
with UV radiation
Formation provides a semiquantitative measure of the degree of rescue
Easily scored by visual inspection

Loss of Function

1. Injection of antisense morpholino oligo

Blocks translation
Injected into fertilized eggs
Morpholinos hybridize to their complementary mRNA
Necessary to have an antibody to the target protein in order to show that the morpholino
has been effective and really prevented protein synthesis

2. Maternal mRNA ablation after injection of antisense deoxyoligonucleotides

Inhibits a maternally acting gene
Injected into the oocyte
 Antisense oligos also hybridizes to the target message
RNA-DNA hybrid is a nuclease target
RNAseH
Destruction is confirmed by RT-PCR
It is necessary to make the treated oocytes into embryos
Maturing the oocytes in vitro with progesterone
Coloring them with a vital dye
Reimplanting them into the abdomen of an ovulating frog

3. Use of dominant negative versions of gene products

Domain-swapped versions of transcription factors
Effector domain of the factor is replaced by a strong activator or a strong
repressor
Strong activator fusion = normal factor activator
Strong repressor fusion = normal factor repressor

No embryonic stem cells

No equivalent method for making knockouts in the mouse by targeted mutagenesis
Zinc-finger nucleases
Cause double-stranded breaks in target loci
DNA repair process generates small insertions or deletions
loss-of-function mutations
Fertilized egg is injected with mRNA encoding the nuclease
Most cells show specific gene ablation with very limited side effects

III. PROCESSES OF REGIONAL SPECIFICATION

Determinants

1. Ventral determinant
Established at oogenesis due to mRNA localization at vegetal cortex
Formation of endoderm
the source of mesoderm-inducing signals forming the mesoderm

2. Dorsal determinant
formation of the organizer at the region of which becomes the dorsal lip
Initially at the vegetal pole but shift towards the dorsal pole due to cortical rotation
Organizer become source of signals (neural plate and pattern the mesoderm)

3. Germ plasm
visible in electron microscope
patch of fibrillar granular material
Contains cat2 mRNA
 express primordial germ cell marker
Appear: mitochondrial cloud (balbani body) of stage II oocyte
End up in: Vegetal cortical region of mature oocytes and early embryo

Evidence of Determinants

1. Twin (Lateral)
Separation of two blastomeres at 2-cell stage
Suggest both halves have determinants

2. Hyperdorsal and Belly piece (Frontal)

Divided frontally (separating prospective ventral and dorsalhalves)
Dorsal halve has both dorsal and ventral tissue

3. Grafting Cytoplasm (direct evidence on the dorsal determinant)

from vegetal pole into UV-ventralized embryos
Components of Dorsal determinant
A. Wnt Signaling pathway
Components of Ventral determinant
A. mRNA encoding the transcription factor vegT.

Early dorsoventral patterning

1. Microtubule elongate in sperm aster

2. Meeting the vegetal cortex

Anchored to cell surface by kinesin
Tubules are aligned with their + ends in the direction of future cortical rotation

3. Rotation Movement
Due to relative displacement of surface-tethered microtubules relative to internal dynein

Cortical Rotation
Dorsal determinant is moved from vegetal pole to dorsal pole
Depends on the array of parallel microtubules, rotation and associated motor protein,
kinesin and dynein

A. Prevention of cortical Rotation

1. Microtubules depolymerizing
2. Injection of Neutralizing Antibodies
3. Vegetal hemisphere is irradiated with UV light before rotation starts

B. Effects on the embryo

1. Radially symmetrical
 2. Extreme ventral in character
3. Mesoderm mainly consist of blood island (normally in ventral midline)

Dorsal Determinant
Consist of component of the conanical Wnt signal transduction pathway

A. Cause stabilization of -catenin

Injection of Wnt mRNA
1. Rescue formation of a dorsal axis in UV-ventralized embryo
2. Induce a second dorsal axis in normal embryo
-catenin
1. Can be seen by immunostaining to enter nuclei in dorsal side
2. Essential role is obtained by antisense oligonucleotide mediated ablation of -catenin mRNA
from oocyte

Treatments:

1. Hyperdorsal embryos
produced by treating early blastulae with lithium salts
resemble radially symmetrical heads

2. UV-ventralized embryos
Arise when mesoderm causes a development as organizer tissue
Rescued to normal pattern by localized injection of lithium

3. Ventral injection
Injection of lithium in the ventral side of a normal embryo will induce a secondary dorsal
axis

4. Mechanism

A. Lithium
Inhibitor of gsk3
Mimics the effects of Wnt signaling
Additional effects through inhibition of the inositol phosphate pathway

B. -catenin
Immediately target Tcf transcription factors

C. Tcf
Converted from an activator to a repressor by association with -catenin
Tcf3 has its repressive activity neutralize

D. Net result
 Upregulation in the dorsal sector organizer genes
1. Encoding many transcription factors
A. Goosecoid
2. Many signaling molecules
A. (BMP) Bone morphogenetic protein inhibitors

VI. INDUCTIVE INTERACTIONS

PROPORTION REGULATION

o If a material is removed from the embryo, the pattern that eventually forms does not have a
gap but rather consists of a reduced-scale version of the normal pattern.

o Pattern is usually not perfect but certainly can tend toward the normal

o Exhibits antiregulative behavior due to the accumulating morphogen at the cut

o Key is the expression of ADMP

o Removal of molecular components that regulate proportion such as BMP 2,4,7 and ADMP
leads to total neutralization

ADMP- antidorsalizing morphogenetic protein; a bone morphogenetic (BMP) type inducing factor

- expressed in the organizer, yet has ventralizing properties

- BMP signaling represses its transcription

ANTEROPOSTERIOR PATTERNING

o Neural-inducing activity is shown both by the organizer and by the axial mesoderm into which the
organizer develops.

o Anterior organizer or anterior axial mesoderm induces brain structures

o Posterior organizer or posterior axial mesoderm induces both brain and spinal cord

o Posterior signal controls anteroposterior patterning during the formation of the central nervous
system (CNS)
 o The two domains within the organizer in the initial anteroposterior pattern are derived from the
different ratio of -catenin arising from cortical rotation and nodal-related signaling arising from
mesoderm induction

Anterior part- will become anterior ectoderm and prechordal mesoderm

*goosecoid will induce expression of anterior-type genes from ectoderm, such as otx2 (fore/ midbrain)
and ag1 (cement gland)

Posterior part- will later become the notochord and somites and, during gastrulation, it elongates
considerably by convergent extension.

*homeobox gene not and the T-box gene brachyury will induce expression of both anterior- and
posterior-type genes from the ectoderm (e.g. both otx2 and Hox genes), and its posteriorizing activity due
to secretion of FGFs and Wnts. Together these upregulate a group of homeodomain transcription factors
encoded by the cdx genes and these in turn upregulate the posterior Hox genes of paralog groups 613.

Evidence that FGF and Wnt signaling is required to induce the trunktail region:

1. If animal caps are treated with the BMP inhibitor noggin, then only anterior-type neural genes are
induced, but addition of Wnt or FGF will also induce posterior neural genes
2. Overexpression in embryos of a dominant negative FGF receptor that inhibits endogenous FGF
signaling, or of the dick- kopf Wnt inhibitor, will prevent formation of the posterior.
3. Overexpression or inhibition of retinoic acid does have effects on the pattern but in Xenopus largely
confined to the hindbrain
FGF- effects mostly felt on the trunk-tail region

Wnt- effects reach on the hindbrain region; controls engrailed [midbrain-hindbrain region]

ORGANIZER GRAFT

o All three of the processes, dorsalization, neural induction, and anteroposterior patterning are
shown here

o A piece of tissue from above the dorsal blastopore lip is implanted into the ventral marginal zone
leading to the formation of a double dorsal embryo.

o The notochord of the ectoderm above the graft becomes induced to form a second neural tube. As
gastrulation proceeds, both host and graft axes form progressively more posterior parts.

o The final result is a symmetrical pair of embryos joined belly to belly.

o The movements of the grafted organizer are autonomous and preprogrammed.

o The secondary axes arising from organizer grafts often lack a head, but if the graft includes the
deep anterior region of the organizer then this will emit cerberus and dickkopf and induce a head in
the overlying ectoderm.