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.

II. NORMAL DEVELOPMENT

III. 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 regio 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 elongted
 ● Induced to form ventral-type tissues
→ swell
→ form transluscent 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

IV. 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 β-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
➔ Β-catenin
1. Can be seen by immunostaining to enter nuclei in dorsal side
2. Essential role is obtained by antisense oligonucleotide mediated ablation of β-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. Β-catenin
● Immediately target Tcf transcription factors

C. Tcf
● Converted from an activator to a repressor by association with β-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 β-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 a​ nd the T-box gene ​brachyury ​will induce expression of both anterior- and
posterior-type genes from the ectoderm (e.g. both ​otx2 a​ nd ​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 6–13.

Evidence that FGF and Wnt signaling is required to induce the trunk–tail 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.