Definitions

Question 1

Xenopus
Q- Xenopus organizer – expression of organizer genes

Answer 1

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).

Question 2

animal hemisphere 3p

Answer 2

The non-yolk-containing (upper) half of the amphibian egg.
During embryogenesis,:
cells in the animal hemisphere divide rapidly
and become actively mobile (“animated”).

Question 3

vegetal hemisphere 4p

Answer 3

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”).

Question 4

cortical rotation

Answer 4

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

Question 5

Cortical rotation – the gray crescent

Answer 5

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

Question 6

Gray crescent:

Answer 6

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.

Question 7

Cortical rotation – microtubule arrays

Answer 7

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

Question 8

Cortical rotation – microtubule arrays
part 2

Answer 8

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

Question 9

CR- Summary +Diagram Slide 17

Answer 9

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.

Question 10

marginal zone

Answer 10

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)

Question 11

vegetal rotation

Answer 11

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.

Question 12

radial intercalation

Answer 12

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)

Question 13

convergent extension

Answer 13

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

Question 14

Polarized cell intercalation drives convergent extension

Answer 14

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

Question 15

What drives convergent extension in the dorsal mesoderm?

Answer 15

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)

Question 16

blastopore

Answer 16

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

Question 17

Nieuwkoop centre

Answer 17

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.

Question 18

The importance of the Nieuwkoop Centre

Experiment

Answer 18

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.

Question 19

Nieuwkoop Centre: dorsal and anterior structures Experiment: Nieuwkoop Centre grafts.

Answer 19

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.

Question 20

In the Nieuwkoop centre
b-catenin and VegT ?

These Nodal-like proteins

Answer 20

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.

Question 21

organizer

Answer 21

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.