Lecture 8- Germ layer formation, axis formation and gastrulation

Question 1

How are the embryonic germ layers formed?

Answer 1

1. Cells derived from the vegetal (lower) hemisphere signal to cells derived from the animal hemisphere
2. Equatorial animal hemisphere cells (in the marginal zone) close to the vegetal hemisphere become mesoderm as they receive signals from the vegetal hemisphere as are closer to it
3. These induced mesodermal cells then begin to signal to part of the ectoderm
4. Animal hemisphere cells further away from the vegetal hemisphere become ectoderm which differentiate into neural tissue
5. The non-responding remainder of the ectoderm becomes epidermis
6. Vegetal hemisphere cells become endoderm

Question 2

What is the evidence that vegetal signals are released and act on equatorial tissue to induce mesoderm?

Answer 2

• Isolated animal cap tissue develops into epidermis and isolated vegetal pole tissue develops into gut-like endoderm
• Tissue explants dissected from the equatorial region between animal and vegetal pole develop into a mixture of ectodermal (epidermis and neural tube) and mesodermal (muscle, blood, mesenchyme and notochord) tissues
• Tissue explants from dorsal and ventral parts of the equatorial regions develop differently
• When animal cap and vegetal pole tissue are combined, then mesoderm is induced in animal cap tissue by the vegetal pole tissue
• Exposure of Xenopus animal cap ectoderm explants to purified TGF-beta signalling molecules (e.g Activin or Xnr proteins induces dorsal axial mesoderm, which exhibits dramatic convergent extension movements)

Question 3

When the unfertilised egg from the animal and vegetal hemispheres?

Answer 3

• The unfertilised egg is polarised such that the yolky vegetal hemisphere contains molecules missing from the animal hemisphere due to gravity
• These localised, yolk-associated molecules are called cytoplasmic determinants

Question 4

What is the difference between the animal and the vegetal hemisphere components?

Answer 4

• The vegetal hemisphere is beige and contains yolky platelets, RNA and protein molecules (cytoplasmic determinants)
• The animal hemisphere is opaque and brown and contains pigment granules

Question 5

What is the role and function of VegT and Nodal after fertilisation?

Answer 5

1. After fertilization and the initial rounds of cell division, vegetally localised VegT mRNA is translated to produce the VegT protein
2. Within these vegetally positioned cells, VegT binds to the transcription regulatory elements of genes that encode Nodel-related morphogens (Xnr genes) and promotes their transcription
3. Nodal-related proteins then diffuse from the VegT expressing cells and into the equatorial region of the embryo

Question 6

What type of molecule is VegT?

Answer 6

A t-box transcription factor and a cytoplasmic determinant

Question 7

What do cells closest to the source of Nodal related proteins become?

Answer 7

Endoderm

Question 8

What do cells intermediate distances to the source of Nodal related proteins become?

Answer 8

Mesoderm

Question 9

What do cells furthest from the source of Nodal related proteins become?

Answer 9

Ectoderm

Question 10

What stage do the germ layers become organised and patterned?

Answer 10

During gastrulation

Question 11

What does the Nieukwoop centre induce?

Answer 11

The formation of the Spemann-Mangold organiser

Question 12

What is the functions of the Spemann-Mangold organiser?

Answer 12

Co-ordinants axis formation and tissue patterning during gastrulation

Question 13

Describe the initial symmetry breaking event which occurs during fertilisation

Answer 13

1. Sperm entry activates cortical cytoplasm
2. The cortical cytoplasm undergoes a 30 degree rotation which redistributes and activates maternal dorzalising factors into the animal hemisphere
3. This movement of vegetal cytoplasm into the animal hemisphere on the opposite side of the embryo to the sperm entry point activates components of the Wnt signalling pathway so high levels of Wnt-induced nuclear beta catenin and Xnr signalling occur simultaneously on the dorsal side
4. This activation is on the dorsal side of the embryo and forms the Nieuwkoop centre

Question 14

How is radial symmetry of the egg broken?

Answer 14

At the point of sperm entry

Question 15

What is the Nieukwoop centre?

Answer 15

A signalling centre on vegetal tissue within the blastula where beta-catenin and nodal are both present

Question 16

How is the Nieuwkoop centre created?

Answer 16

1. Cortical rotation of vegetal cytoplasm upwards through the equator is accompanied by translocation of Wnt11 mRNA and dishevelled protein
2. Wnt11 mRNA and dishevelled protein are transported along microtubules towards the animal pole on the future dorsal side of the embryo
3. After several cleavage divisions, activated β-catenin accumulates in the nuclei of cells on the future dorsal side of the embryo, causing transcription of Wnt-pathway target genes
4. Canonical Wnt signalling causes the accumulation of β-catenin transcription factor in the nuclei of target cells
5. These target genes include the Nodal-related genes
6. The presence of β-catenin on the dorsal side then increases Xnr gene expression and creates a gradient of Nodal-related proteins underneath the developing mesoderm

Question 17

What molecules induce the formation of the Spemann-Mangold organiser?

Answer 17

• The combined action of VegT and Beta-catenin within the Nieuwkoop Centre on the dorsal side of the embryo promotes high enough levels of Nodal-related protein signalling to induce the Spemann-Mangold Organiser in the dorsal mesoderm.
• Wnt signalling stablises and promotes the nuclear localisation of beta-catenin

Question 18

Describe an experiment which provides evidence for the existence of the Nieuwkoop centre

Answer 18

o Take a dorsal vegetal blastomere from an early blastomere stage, from one embryo and transplant into the ventral side of a host embryo

o The embryo develops as a twinned embryo with a duplicated axis

Question 19

What proteins produce dorsal mesoderm character and what proteins produce ventral mesoderm character?

Answer 19

Dorsal mesoderm: Nodal/Xnr proteins and Wnt signalling

Ventral mesoderm: BMPs

Question 20

How is ventral mesoderm character created?

Answer 20

A gradient of BMP signals is created and sustained in the ventral mesoderm of gastrula stages by a ventrally located protease that specifically destroys chordin proteins, allowing BMPs to accumulate in ventral tissue

Question 21

What happens if Nodal related proteins are low?

Answer 21

Ventral mesoderm is induced

Question 22

How are Spemann-Mangold organiser genes transcribed (chordin, noggin, goosecoid) from the induction of beta-catenin and Xnr proteins?

Answer 22

• Beta-catenin induces siamois gene transcription
• Xnr proteins induce Smad2 protein activation
• Siamois and Smad2 transcript chords, noggin and goosecoid

Question 23

What DNA binding transcription factors does Xnr and beta-catenin induce?

Answer 23

• Beta-catenin induces siamesi gene transcription

* Xnr proteins induce Smad2 protein activation

Question 24

How does the Spemann-Mangold organiser initiate gastrulation?

Answer 24

The targets of Organiser-specific transcription factors in combination with the Brachyury targets creates dorsal axial mesoderm which initiates the gastrulation process•

Question 25

Where is goosecoid, Not1, Lim1 and brachyury transcription factors expressed?

Answer 25

• Goosecoid, Not1 and Lim1 are specifically expressed in the organiser
• Brachyury is expressed throughout the mesoderm including the organiser

Question 26

Where is the organiser first visible/where is gastrulation initiated?

Answer 26

Dorsal blastopore lip

Question 27

The Spemann-Mangold organiser patterns the embryos A/P and D/V axes by regulating what?

Answer 27

1. Initiating and co-ordinating involution, intercalation and migration of dorsal axial mesoderm over the inner surface of the blastocoel roof as part of a process called convergent extension
2. The production of multiple distinct fates in the correct positions and proportions within the mesoderm: prechordal mesoderm, notochord, paraxial mesoderm (somites), intermediate mesoderm, lateral plate mesoderm and blood islands

Question 28

The organiser is a dynamic mix of axial mesodermal progenitor cells that give rise to what three distinct embryonic tissues?

Answer 28

Anteriorly migrating pharyngeal endoderm, prechordal mesoderm and notochord

Question 29

What is the blastopore lip and the organiser called in vertebrates?

Answer 29

Blastopore lip = primitive streak

Organiser = Henson’s node

Question 30

What is the direction of cell movement in gastrulation?

Answer 30

From posterior to anterior