Definitions Flashcards
Xenopus
Q- Xenopus organizer – expression of organizer genes
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. (This is the first mention of the organizer – more on this later).
animal hemisphere 3p
The non-yolk-containing (upper) half of the amphibian egg.
During embryogenesis,:
cells in the animal hemisphere divide rapidly
and become actively mobile (“animated”).
vegetal hemisphere 4p
The bottom portion of the amphibian egg,
containing yolk, which serves as food for the developing embryo.
The yolk-filled cells are divide more slowly and
undergo less movement during embryogenesis (and hence are like plants, or “vegetal”).
cortical rotation
following sperm entry the outer layer (the cortex) of the egg loosens from the inner dense yolky core
outer layer (the cortex) includes the plasma membrane of the egg, cytoskeletal components, rough ER and other components
following loosening there is a yolk free area between the core and cortex called the shear zone
midway through the 1st cell division the cortex begins to rotate relative to the core (this is CORTICAL ROTATION) – continues to near end of 1st cell cycle
cortical rotation – results in a ~ 30° displacement of the vegetal cortex away from sperm entry site towards the future dorsal region
cortical rotation coincides with the translocation of a maternal “dorsalizing activity” from the vegetal pole to the future dorsal side of the embryo
Cortical rotation – the gray crescent
midway through the 1st cell cycle arrays of parallel microtubules (MTs) appear in the vegetal shear zone
both cortical rotation AND the translocation of dorsal determinants depend on these parallel arrays of MTs
as a result of cortical rotation a back of inner gray cytoplasm can sometimes be observed – called the gray crescent
Gray crescent:
A band of inner gray cytoplasm that appears following a rotation of the cortical cytoplasm with respect to the internal cytoplasm in the marginal region of the 1-cell amphibian embryo. Gastrulation starts in this location.
Cortical rotation – microtubule arrays
parallel arrays of microtubules form –with plus ends oriented away from the site of sperm entry
during cortical rotation the cortex and the “dorsalizing activity” move towards the plus ends
destruction or depolymerization of MTs (by uv light, nocodazole treatment or injection of antibody that inhibits microtubule associated protein called XMAP230) results in embryos lack all dorsal-anterior structures
without cortical rotation the “dorsalizing activity” remains stuck in the vegetal region where it is not active
Cortical rotation – microtubule arrays
part 2
the subcortical vegetal region cytoplasm of embryos treated to prevent microtubule array formation is still effective in inducing dorsal axis in cytoplasmic transplantation experiments
THEREFORE, dorsal specification requires not just the dorsalizing activity, but its translocation from the vegetal region towards the marginal zone (area between the animal and vegetal hemispheres)
Implicit in these observations is that the “dorsalizing activity” must be relocated in order to become activated
CR- Summary +Diagram Slide 17
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. (This is the first mention of the organizer – more on this later).
Cortical rotation is more subtle and occurs after the realignment to gravity.
marginal zone
marginal zone (area between the animal and vegetal hemispheres)
(involuting marginal zone = IMZ= dorsal side)
Continued epiboly helps push chordamesoderm into involuting marginal zone.
Marginal zone: In amphibians: Where gastrulation begins, the region surrounding the equator of the blastula, where the animal and vegetal hemispheres meet.
the animal cap and noninvoluting marginal zone cells expand by epiboly to cover the entire embryo and will become the surface ectoderm
the involuting marginal zone (IMZ) undergoes radial thinning (epiboly) pushing tissue into dorsal lip
the post-involution marginal zone extends via medial lateral intercalation of cells = convergent extension
the involuting marginal zone (IMZ) and the noninvoluting marginal zone (NIMZ) are initially several cell layers deep and become one thin broad layer (radial intercalation)
marginal zone will become the internalized mesoderm and endoderm
- Gastrulation
equatorial cells (marginal zone between animal and vegetal hemispheres) become progenitors of mesoderm (bone, muscle, heart)
vegetal rotation
Vegetal rotation in endoderm, places mesoderm
Vegetal rotation: During frog gastrulation, internal cell rearrangements place the prospective pharyngeal endoderm cells adjacent to the blastocoel and immediately above the involuting mesoderm.
Gastrulation is initiated by two movements epiboly and vegetal rotation
Vegetal rotation moves endoderm animal and dorsal
While epiboly pushes ectoderm vegetal, vegetal rotation lifts IMZ cells into interior. This places anterior endoderm (pharynx) in the correct position above anterior mesoderm. Continued epiboly helps push chordamesoderm into involuting marginal zone.
2 hours before bottle cells are observed, cell rearrangements on the dorsal floor of the blastocoel – cells pushed up towards animal cap = vegetal rotation
Vegetal rotation driven by cell intercalations (A)
Notice that epiboly pushes ectoderm vegetal while vegetal rotation pushes neighbouring mesoderm animal and the dorsal lip forms at this junction via invagination
Vegetal Rotation and involution at dorsal lip
Important point is dorsal lip formation, initial mesoderm involution requires vegetal rotation and epiboly
Go back to vegetal rotation, it puts pharyngeal endoderm up against dorsal side of embryo
Those cells bind ECM (FN - FIBRONECTIN) and by pulling guide cells to anterior. If you block that interaction gastrulation fails.
radial intercalation
Epiboly driven by radial cell intercalation
Epiboly driven by chemo-attractant that pulls deep cells towards superficial layer (cell intercalation)
Vegetal rotation driven by cell intercalations (A)
As cells intercalate medial and dorsal (B) tissue is moved animal (C).
epiboly (in this case) also involves increase in cell number (cell division = mitosis) in addition to the concurrent integration of several deep layers into one (radial intercalation)
As pre-involution mesoderm approaches dorsal lip it also undergoes medial lateral intercalation
the post-involution marginal zone extends via medial lateral intercalation of cells = convergent extension
Polarized cell intercalation drives convergent extension
the involuting marginal zone (IMZ) and the noninvoluting marginal zone (NIMZ) are initially several cell layers deep and become one thin broad layer (radial intercalation)
convergent extension
Convergent extension in pre/post involution mesoderm (involuting marginal zone = IMZ= dorsal side)
the post-involution marginal zone extends via medial lateral intercalation of cells = convergent extension
Polarized cell intercalation drives convergent extension
Post-involution mesoderm lies next to blastocoel roof and will extend towards anterior via convergent extension
when cells pass around the blastopore lip – they involute and undergo convergent extension along mediolateral axis – resulting in a long narrow band of mesoderm
Polarized cell intercalation drives convergent extension
polarized cell cohesion –cells send out protrusions to contact one another – protrusions are not random but are directed toward midline of embryo
Requires cadherin based adhesion and actin myosin contractility
Polarization of post-involution mesoderm cells is active process lying downstream of wnt signalling
What drives convergent extension in the dorsal mesoderm?
polarized cell cohesion (as previously discussed) – involuted mesoderm cells send out protrusions to contact one another – protrusions are not random but are directed toward midline of embryo and this requires extracellular layer of fibronectin
differential cell cohesion – cell type specific expression of different cadherins causes cell types to sort into different groups (i.e. notochord and paraxial mesoderm)
calcium flux – waves of intracellular calcium and contraction sweep across dorsal tissues undergoing convergent extension (regulated actin filament contraction)
blastopore
cells invaginate to form a slit-like blastopore – often called blastopore “lip”
Edges of blastopore spread ventrally, become circle
Mesoderm involution occurs all around blastopore lip but to much lesser extent than dorsal side
mesoderm entering the dorsal lip becomes prechordal mesoderm (heart, pharynx) and the dorsal mesoderm (notochord* and somites**)
mesoderm entering the more lateral areas of the blastopore will form the lateral plate mesoderm (kidneys, bones and other organs)
for whatever reason – the patch of endodermal cells visible within the ring of the blastopore lip is called the yolk plug (this is not a mass of yolk, but cells enriched in yolk content)
internalization of the mesoderm and endoderm starts at the blastopore
mesoderm and endoderm converge and begin to move inwards at dorsal lip of the blastopore
In amphibians, the dorsal lip cells of the blastopore and their derivatives (notochord and head endomesoderm).
fate mapping shows that gray crescent area becomes cells that form the dorsal lip of the blastopore
a self-determining tissue - THE DORSAL LIP of the blastopore – derived from the region of the gray crescent cytoplasm
this one special region - THE DORSAL LIP of the blastopore – derived from the gray crescent cytoplasm – was named “the ORGANIZER”
a group of cells of the early amphibian embryo (the dorsal blastopore lip) derived from the region of the embryo where the gray crescent was observed at the time of cortical rotation
Nieuwkoop centre
the dorsal most vegetal cells of the blastula can induce formation of an “organizer” – these cells are known as the Nieuwkoop centre
In Xenopus, before fertilization, the egg has a radial symmetry.
Sperm entry sets up the D/V axis with the dorsal side of the embryo situated at a place opposite of the sperm entry point.
Within the first 90 minutes after fertilization, the cortex rotates approximately 30 degrees.
The vegetal cortex opposite the sperm entry point moves towards the animal pole.
This leads to the formation of a ‘signaling centre’ opposite the site of sperm entry known as the Nieuwkoop Centre.
This process leads to bilateral symmetry with the Nieuwkoop Centre at the midline separating right and left sides of the embryo.
The importance of the Nieuwkoop Centre
Experiment
The importance of the Nieuwkoop Centre
The Nieuwkoop Centre sets up D/V polarity in the blastula and is essential for normal development. The 1st cleavage cuts through the site of sperm entry and the Nieuwkoop Centre. The 2nd cleavage splits the embryo into 4 cells (2 with the Nieuwkoop Centre and 2 without).
Experiment: Divide the 4 cell embryo into two halves, one dorsal and one ventral. The half with the Nieuwkoop Centre develops fairly normally missing only some ventral structures. The half without the Nieuwkoop Centre develops very abnormally to produce a radially symmetric embryo without dorsal or anterior structures.
Nieuwkoop Centre: dorsal and anterior structures Experiment: Nieuwkoop Centre grafts.
Nieuwkoop Centre: dorsal and anterior structures Experiment: Nieuwkoop Centre grafts. 1) When cells of the Nieuwkoop Centre are grafted onto the ventral side, twinned embryos with two ‘dorsal sides’ are produced. 2) When cells of the ventral side are grafted onto the dorsal side, normal embryos are produced (no effect). Therefore, signal from the Nieuwkoop Centre must be required for developing dorsal structures.
In the Nieuwkoop centre
b-catenin and VegT ?
These Nodal-like proteins
In the Nieuwkoop centre
b-catenin and VegT transcription factors activate the expression of Nodal-like proteins.
These Nodal-like proteins are paracrine factors (signaling proteins of the TGFb family) secreted from the Nieuwkoop centre.
organizer
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. (This is the first mention of the organizer – more on this later).
The rest of this lecture set mostly concerns the region of the amphibian embryo known as “the organizer”
This was a major advance in the field of embryology
the discovery of the organizer
how the organizer is formed
the function of the organizer
OVERALL: We will be see how the concept of the organizer was developed by classical methods through manipulating amphibian embryos, and how modern molecular biology has now defined the organizer in terms of genes and gene expression.
