Xenopus

Question 1

Descirbe the Xenopus egg and the cleavange divisions it undergoes.

Answer 1

The Xenopus egg consists of a dark pigmented animal hemisphere and light pigmented vegetal hemisphere, with a (equitorial) marginal zone. The fertilised egg undergoes holiblastic* cleavage and by 7 hours has formed a 5000 cell blastula, with a fluid fill blastoceol in the animal hemisphere. Gastrulation occurs after 10 hours with the appearance of the doral blastopore lip in the equatorial region of the vegetal hemisphere. Maternal-zygotic transition occurs at the blastula stage. Gastrulation requires the zygotic genes.

Question 2

How were fate maps of the amphibian gastrulae made and what do they show?

Answer 2

They were genereated by labelling cells of the amphibian gastrulae with vital dyes and looking at what tissues they formed in tadpoles.

• Animal pole –> ectoderm
• Marginal zone –> mesoderm
• Vegetal pole –> endoderm

Question 3

What is the dorsal blastopore lip?

Answer 3

The dpl is formed by bottle cells that undergo apical constriction. The dpl expands around the enitre circumference of the embryo before closing over the vegetal hemisphere. Closure of the dpl indicates the end of gastrulation.

Question 4

Describe the important poiints of Xenopus gastrulation

Answer 4

Gastrulation is a phase of morphogenetic movements in which the marginal zone (a belet of tissue around the equator) becomes internalised through an opening called the blastopore. The start of gastrulation is marked by the dpl which is formed of bottle cells. During gastrulation mesoderm and endoderm involute into the blastoceol and migrate along the inner surface of the animal hemisphere. A new cavity is created (the archeneron) that will become the lumen of the gut. The blastopore forms the anus while the mouth forms when the anterior endoderm fuses with the ectoderm. By the end of gastrulation the animal cap ectoderm covers the entire external surface of the embryo and the yolky vegetal tissue has become a mass of endoderm in the interior.

Question 5

Describe neural tube closure.

Answer 5

At the end of gastrulation, the nervous system is a flat simple epithelial sheet on the dorsal side of the embryo, called the neural plate. The lateral edges of the neural plate elevate and fold inwards where they fuse in the dorsal midline forming the neural tube.

Question 6

Describe the amphibian body plan that is shared by all cordates.

Answer 6

See figures on slides for confirmation.

Question 7

What did Spemman do in 1909?

Answer 7

1. Seperated an embryo at the 2 cell stage using a fine hair. Both cells developed into two normal but half size embryos.
2. He also seperated a gastrulae using the dorsal blastopore lip as a marker, obtaining a ventral and dorsal half. The dorsal half found a normal, half-sized embryo, whereas the ventral half formed a ball of epidermis and blood (ventral mesoderm) Thus the dpl is needed for regulative development (and dorsal tissue).

Question 8

What did Holtfreter do (1933)?

Answer 8

Divided the embryo into smaller fragments to see how they developed in vitro (aka. specification).

• Cells taken from the animal hemisphere differentiated into epidermis
• Cells taken from the vegetal hemisphere don’t differentiate
• Cells from the dorsal marginal zone differentiate into largely notochord (and some muscle)
• Cells from the ventral and lateral segments formed blood and mesothelium

Question 9

What can be understood when comparing a fate map and specification map of the early Xenopus embryo (gastrulae)?

Answer 9

Most of the nervous system, somatic tissues, the heart and pronephros are not specified. Cells that are fated to form these tissues in the fate map form ventral tissues (epidermis, blood, mesothelium) in the specification map. The notochors is the only dorsal tissue that is specified in the early gastrulae.

Question 10

What did speman do in 1919 and what has been found out from his experiments?

Answer 10

1. He used to different species (triturus was one) to carry out a transplant. He replaced ventral ectoderm (epidermis) with dorsal ectoderm (neural plate) and found that dorsal ectoderm always formed epidermis if transplanted at the beginning of gastrulation but neural plate if transplanted at the end of gastrulation. He concluded that the nervous system is specified during gastrulation.
2. The reverse experiment was also carried out. Replacing dorsal ectoderm with ventral ectoderm. Analgous results were found. Therefore it was concluded that epidermis must also be specified during gastulation.

Question 11

What did Spemann and Mangold do in 1924? (One of the most famous embryological experiments!)

Answer 11

Transplanted the dpl of an early gastrulae to the ventral side of equivalent staged host.

• A second axis was formed
• The notocord came from graft tissue
• Neural tube, somites, and kidney was predominately formed by host tissue that would usually have formed ventral mesoderm (blood and mesothelium) and ectoderm (epidermis)
• The graft managed to respecify ventral tissue to form a second dorsal axis
• The dpl was called the organiser

Question 12

What are the gastrula organisers called in the other model systems?

Answer 12

• Amphibian - dorsal blastopore lip
• Zebrafish - the shield
• Chick - Henson’s node
• Mammal - node

Question 13

What did Holtfreter do in 1933?

Answer 13

Incubated Triturus gastrulae in a high salt solution and found that the blastocoel collapsed. The mosoderm and endoderm exogastrulated, creating an ectodermal that failed to produce neural tissue. As the ectodermal cap is still connected to the ograniser the planar route is still viable, however the vertical route is not as there is no underlying mesoderm. It was concluded tht verticle signals from the mesoderm are required for neural induction.

Question 14

What experiments casted doubt on the specificty of the organiser?

Answer 14

1. Animal caps (isolated by high salt solution) could be induced to form neural tissue by a vast range of chemicals.
2. Dead organisers (killed by heat, freezing, drying or fixing in alcohol) placed in the belly of the embryo underlying ventral ectoderm could induce neural tissue
3. Even a grain of sand induced neural tissue! (tested by Sox3 - a neural tissue marker)
4. Barth 1941 - a chnage in pH could induce animal caps to forma neural tissue.
5. Holtfreter 1944 - caps resonded to mild dissociation to form neural tissue.

Stress is sufficient to induce neural tissue in the animal cap. Animal caps are primed to form neural tissue but are under repression. The organiser blocks this repression.

Question 15

Describe what is known about Chordin and how it was identified.

Answer 15

• It was not untill the molecular era that molecules/genes in the spemann’s organiser could be identified. They were first identified using WISH and probes made from RNA isolated from the early gastrula dorsal blastopore lip. Many transcription factors were found but these were not going to be the signal?? The gene chordin which encoded a secreated protein was the first to be identified.
• Injection of Chordin mRNA into ventral blastomeres induced a cojoined twin
• Chordin can mimic the Spemann organiser
  *

Question 16

How was Noggin identified and what learnt of it?

Answer 16

• Made a clonal library of all the mRNA found in the organiser. Chose groups of colonies (pools) and injected into an embryo that had the organiser destroyed by UV radtion. Assayed to see what colony pools rescued the dorsal development. These pools are then seperated into smaller pools and eventually individual mRNA.
• The secreted protein Noggin was found to induces a second dorsal axis

Question 17

1. What is the order of dorsalising strength of the molecules, Noggin, Chordin and Follistatin?
2. What is most abundantly expressed?

Answer 17

1. Noggin–>Chordin–>Follistatin
2. Chordin–>Noggin–>Follistatin

• Noggin, Chordin (and follistatin) could not induce the most rostral tissues when injected into the embryo
• N, C and F all induces neural tissue when added to animal caps, including neural tissue of rostral character.

Question 18

How do morpholinos lead to knock-down of gene expression?

Answer 18

The bind to complementary sequences in the 5’ UTR or the intron-exon boundary. The former blocks translation and the latter blocks splicing (to produces a truncated protein)

Question 19

What was found when Chordin was blocked by AMOs?

Answer 19

Two AMOs were used to knock-down Chordin, because xenopus laevis is tetraploidy. When injected together the AMOs caused the embryos to have greatly reduced heads. The notocord is missing and the ventral blood islnd expnded. Thus the embryo was said to be ventralised. The phenotype was injected by Chordin mRNA that was not target by the AMOs.

Question 20

What was found when Chordin AMOs injected organisers were grafted to the ventral ectoderm of a host?

Answer 20

The Chordin AMOs injected organisers did not induce a secondary axis. Therefore, Chordin is necessary for the dorsalising and neuralising activity of a transplanted organiser.

Question 21

What was found from injecting combinations of Noggin, Chordin and Follistatin AMOs?

Answer 21

Only injections containing Chordin AMOs had any developmental effects. When all three AMOs were injected together there was complete loss of the nervous system, notocord and myotome, and expansion of ventral tissues. Shows that all the genes are acting together to create the dorsal half of the embryo.

Question 22

How did SOG shed new light onto the dorsal ventral patterning of the xenopus?

Answer 22

SOG is a similiar structure to Chordin found in drosophila. A lot was already known about SOG. Wanted to see if SOG had the same effects as Chordin and whether this knowledge could then be extrapolated to Chordin.

• When SOG mRNA is injected into the ventral embryo a secondary axis is formed (like seen in Chordin)

Question 23

What is BMP?

Answer 23

• Bone morphogenetic proteins
• Originally found in protein extracts that form ectopic bone/cartilage formation in rodents
• Part of the TGF-B superfamily

Question 24

Describe the basic BMP signalling pathway

Answer 24

BMP ligand (2,4,7) binds to type I (RI) and type II (RII) receptors. Binding allows RII to phosphorylate RI. RI then phosphorylates SMAD1 which in turn can now bind to SMAD4. This complex enters the nucleus and acts as a cofactor to regulate transcription of target genes

Question 25

What has happens to BMP expression in the early embryo?

Answer 25

• No expression in the organiser in the early gastrulae and the neural plate in the late gastrula.
• Expression is restricted to ventral and lateral areas
  *

Question 26

What does injection of BMP mRNA, AMO and dominant-negative show? What happens when adding BMP to animal caps?

Answer 26

1. Ventralisation of tissues
   
   • Phenotype is identical to embryos lacking the organiser
   • Organiser-specific genes (Chordin) is initially expressed but then switched off in the late gastrulae
2. Mild dorsalising effects
   
   1. Stongest phenotype when all three AMOs (BMP2,4,7) are injected - suggests overlapping function
3. Induces a cojoined twin
   
   1. Kinase domain of BMP RI is deleted. It can still bind to BMP and form complexes with RII. Therefore reducing normal protein function
4. Differentiate as epidermis
   
   1. Indicates that BMP4 is a an epidermalising factor
   2. And that default pathway of ectoderm is neural tissue

Question 27

What are Chordin, Noggin and Follistatin in relation to BMP?

Answer 27

Dominant inhibitors. They bind to BMP and prevent it from binding to its receptor.

Question 28

Describe the bigger picture seen in the modulators of BMP Signalling.

Answer 28

• Patterning of the DV axis is very complicated with many molecules involved.
• “Simplified” see saw model
  
  • Dorsal (crescent) and ventral (Sizzled) inhibitors of tolloid metalloproteases adjust the DV gradient of BMP signalling through cleavage of the BMP inhibitor Chordin.
  • Draw the model