BIOL 303 Study Questions Set 8 Part II and Set 9
1. What distinguishes the animal pole from the vegetal pole of the Xenopus egg?
Visually, animal pole is dark and vegetal pole is light.
Animal pole = upper part of egg, no yolk. During embryogenesis, cells divide
Vegetal pole = lower part of egg, yolk. Serves as food for developing embryo and
cells divide more slowly.
2. At what stage of meiosis is the Xenopus egg at the time of fertilization? Where on
the egg does fertilization occur?
Fertilization can occur anywhere on the animal hemisphere. Point of sperm entry
determines orientation of dorsal and ventral axis (point of sperm axis will be
3. What is the grey crescent and how does it arise? What is the destiny of the grey
Sperm centrioles organize the microtubules in the egg. Microtubules in vegetal
pole are arranged parallel to one another. The microtubule tracks allow for the
cortical cytoplasm to rotate (30 degrees) within the inner cytoplasm. The cortical
rotation can reveal a gray band of inner cytoplasm called the gray crescent.
A rotation of the cortical cytoplasm 30 degrees with respect to the internal
cytoplasm in the marginal region of the 1-cell amphibian embryo.
The cortical rotation breaks the radial symmetry of the amphibian egg, specifying
the orientation of the embryonic body axes. The entire outer cortex of the
fertilised egg rotates relative to the mass of inner cytoplasm by an angle of about
30° about an axis perpendicular to the primary animal-vegetal axis. As a result of
this cortical rotation, 'dorsal determinants', factors able to trigger the formation of
the 'organiser' region of the gastrula, are displaced from the vegetal pole region to
a more equatorial position where they become activated.
Gastrulation starts here. 4. What pattern of cleavage is observed in the Xenopus embryo?
Radially symmetric and holoblastic
First two cleavage planes are meridonial (1 bisects the gray crescent), 3 is
equatorial – but displaced towards animal pole – this makes four smaller animal
blastomeres and four larger vegetal blastomeres
The second cleavage may actually start in animal pole before first is completed in
the vegetal pole
Cleavage continues for 12 cycles.
Results in many smaller cells in animal pole than vegetal pole due to yolkiness in
Becomes morula when 16 64
Becomes blastula at 128 cells
5. What purposes might we attribute to the Xenopus blastocoel?
Permits cell migration during gastrulation
Prevents early cell-cell interactions
6. When does the mid-blastula transition occur and what molecular events are
associated with zygotic gene activation of Xenopus?th
Mid-blastula transition occurs late in the 12 cycle (last cycle)
Gene transcription and cell cycle adds G phases
Blastomeres become motile
DNA demethylation (promoters are demethylated so it promotes transcription)
and histone methylation (associated with active transcription)
7. What are the first signs of gastrulation in Xenopus?
Purpose of gastrulation: bring some cells inside to form endoderm, leave some on
the outside to form the ectoderm, and get some inbetween to form the mesoderm.
In Xenopus, mesoderm is derived from deep blastula layers and endoderm and
ectoderm are derived from more superficial layers.
Gastrulation initiates on future dorsal side of embryo.
Cells invaginate to form a slit-like blastopore. The invaginating cells change
shape to become bottle cells.
Vegetal rotation: 2 hours before bottle cells are observed, cell rearrangements on
the dorsal floor of the blastocoel – cells pushed up towards animal cap
Involution of the marginal zone cells. The animal cells undergo epiboly (thinning
and spreading of layers) and converge and blastopore (opening into archenteron –
cavity forming primitive gut).
Migrating marginal cells turn inward at dorsal lip and travel along blastocoel roof.
First cells to pass over the lip are prospective pharyngeal endoderm (forms the
salivary glands, thyroid gland, pharyngeal pouches). Next is prechordal plate
(forms head mesoderm) and then chordamesoderm (forms notochord – induces
developmental patterns in nervous system). 8. Draw a diagram of the cross section of the early Xenopus gastrula. Label the
blastocoel, archenteron, and blastopore.
9. What are some of the molecular events associated with convergent extension?
What drives the movement during gastrulation? Cells in the involuting marginal
zone and the noninvoluting marginal zone that are initially several cell layers deep
become one thin broad layer. This is called radial intercalation.
When the deep cells pass around the blastopore lip, they involute and undergo
convergent extension (cell movement and tissue elongation) along mediolateral
axis. This results in a long narrow band of mesoderm.
Polarized cell adhesion: – involuted mesoderm cells send out protrusions to
contact one another – directed toward midline of embryo and depend on
extracellular layer of fibronectin
Differential cell cohesion – cell type specific expression of different cadherins
Calcium flux – waves of intracellular calcium surge across dorsal tissues
undergoing convergent extension (regulates actin filament contraction) 10. The Xenopus egg is already polarized at the time of fertilization. In which axis is
the unfertilized egg polarized? An in situ hybridization of what transcript attests to
the above statement?
The unfertilized egg is polarized in the animal-vegetal pole axis.
Vegetal cells determine the body formation plan. The mRNA of transcription
factors VegT is anchored to cortex of vegetal hemisphere. They differentiate to
form the endoderm and induce cells above them to become the mesoderm. VegT
activates transcription of TGF-β related genes (paracrine factors required for
mesoderm induction). By knocking out VegT transcripts, the embryo develops all
Xenopus tropicalis VegT. whole-mount in situ hybridization analysis of tVegT
mRNA expression in tropicalis oocyte. tVegT mRNA is localized to the vegetal
half of a stage VI oocyte.
In Xenopus A/P is linked with the establishment of D/V – and this is set up by
events occurring at fertilization
Dorsal is established opposite point of sperm entry
Subsequent movement of involuting mesoderm sets up the A/P axis
Inductive interactions: the first endomesoderm moves around the dorsal lip
induces ectoderm above it to form anterior structures
Inductive interactions: later involuting mesoderm induces ectoderm above it to
form posterior structures
11. Transplantation experiments involving early and late gastrulation give
dramatically different results. What is the general explanation for these
observations – or what concept was developed from these types of experiments?
Early gastrula: presumptive neural ectoderm transplanted to site of presumptive
epidermis resulted in epidermis therefore conditional, regulative
Late gastrula: presumptive neural ectoderm transplanted to site of presumptive
epidermis resulted in two neural plates therefore autonomous, mosaic 12. What is the Xenopus “organizer” (aka Spemann-Mangold organizer) and how
was it discovered?
This process, whereby the central nervous system (ectoderm) forms through
interactions with the underlying mesoderm, has been called primary embryonic
induction and is one of the principal ways that the vertebrate body becomes
organized. Its discoverers called the dorsal blastopore lip and its descendants “the
Organizer,” and found that this region is different from all the other parts of the
Spemann’s Organizer: More correctly, the Spemann-Mangold Organizer. In
amphibians, the dorsal lip cells of the blastopore and their derivatives (notochord
and head endomesoderm). Functionally equivalent to Hensen’s node in chick, the
node in mammals, and the shield in fish. Organizer action establishes the basic
body plan of the early embryo.
Ligation experiments: demonstrated nuclear equivalence. The first ligature was
set up in the same plane as the first division (plane of cytokinesis) 2 normal
Alternate ligation experiment: similar experiment but the ligature was set up
perpendicular to the plan of cytokinesis 1 normal embryo and 1 bauchstuck
developed (ball of unorganized ventral cells [ectoderm, mesoderm, endoderm], no
dorsal structure [nervous system, notochord, somites])
Ligature experiments proved that something about the grey crescent is critical for
proper development. They also found one special region of the amphibian region had an autonomous
fate: a self-determining tissue – the dorsal lip of the blastopore – derived from the
gray crescent cytoplasm – named the organizer.. Transplantation of dorsal lip
tissue into recipient belly skin region had the remarkable effect of initiating
gastrulation and embryogenesis in surrounding tissue.
13. What is the Nieuwkoop centre?
In Xenopus embryos, the Spemann-Mangold organizer establishes the dorsal-
ventral and anterior-posterior axes. According to the prevalent model of early
development, the organizer is induced by the dorsalizing Nieuwkoop signal,
which is secreted by the Nieuwkoop center. Formation of the center requires the
maternal Wnt pathway, which is active on the dorsal side of embryos. Nieuwkoop
center produce dorsalizing signals in vitro.
After the sperm entry (determines D/V axis