Lecture 9- Axis formation, neural induction and neural tube patterning

Question 1

Define gastrulation

Answer 1

Is the process by which mesoendodermal progenitors move from the blastopore lip to the interior of the embryo

Question 2

By which pathway do organiser precursor cells move?

Answer 2

Through the fibronectin rich pathway across the roof of the blastocoel

Question 3

What cells does the organiser comprise of and what 3 distinct embryonic tissues do these give rise to?

Answer 3

Organiser comprises axial mesoendodermal progenitor cells that give rise to:

1. Pharyngeal endoderm
2. Prechordal mesoderm
3. Notochord

Question 4

Which process defines the A/P axis of the embryo?

Answer 4

Gastrulation

Question 5

Outline the Spemann-Mangold organiser grafting experiment

Answer 5

1. Dorsal blastopore lip was removed from one early gastrula stage newt embryo and transplanted into the ventral side of a second newt embryo
2. Both the host dorsal lip and transplanted dorsal lip contributed to a twinned embryo with two neural tubes and sets of dorsal axial mesodermal tissues- notochord and somites
3. Interestingly, the grafted dorsal lip only contributed to small portions of the second neural tube and axial mesodermal structures, indicating that most of the second axis had been induced by the translated tissue in the host tissue i.e the host tissue fate has been reprogrammed
4. The dorsal lip was thus called the organiser because of its ability to induce new patterns of development and differentiation in surrounding tissue

Question 6

How were molecules with Spermann-Mangold organiser activity discovered?

Answer 6

• By identifying mRNAs that were capable of inducing the formation of a second embryonic axis when injected into the Xenopus embryo
• Discovery of neural inducing activity of mRNAs expressed in the Organiser: injected RNA into animal pole of fertilised egg, dissect animal cap tissue and culture in vivo
• Instead of developing into epidermis, the tissue differentiated into neural tissue (as secreting noggin mRNA which changes the tissues fate)

Question 7

What is the outcome if a grafted organiser was taken from the dorsal blastopore lip of an early gastrula donor and transplanted to an early gastrula host?

Answer 7

Then a complete second axis including head and trunk developed

Question 8

What is the outcome if a grafted organiser was taken from the dorsal blastopore lip of an late gastrula donor and transplanted to an early gastrula host and what does this suggest about the inductive properties of the organiser?

Answer 8

Then only a partial second axis with a portion of trunk but no head developed

Evidence that the inductive properties of the organiser changes during gastrulation

Question 9

Outline the movement of tissue at the different gastrula stages

Answer 9

1. In the early gastrula, the dorsal lip tissue begins to migrate over the blastocoel roof
2. By the mid-gastrula stage, the early dorsal lip tissue has moved deep into the interior of the embryo- this is the tissue fated to become pharyngeal endoderm and prechordal mesoderm
3. Back at the dorsal blastopore lip, a new set of cells begins its journey which will become the posterior of the notochord

Question 10

Why do organiser grafts taken from different staged donors have different inducing potentials?

Answer 10

Because the organiser tissues express different combinations of signalling molecules

Question 11

What are the 4 related functions of the Spemann-Mangold organiser?

Answer 11

1. It creates the A/P axis of the embryo
2. It indues neural tissue from ectoderm
3. It patterns this neural tissue whilst creating the A/P axis of the embryo
4. It introduces A/P and D/V patterns into the mesoderm

Question 12

What molecules are secreted from the pharyngeal endoderm and what do they inhibit?

Answer 12

Releases Dickkopf, Frzb, Tiki which inhibits Wnt signalling and Cerberus which inhibits BMPs

IGF is also secreted which inhibits both Wnt and BMPs

Question 13

What molecules are secreted from the prechordal plate mesoderm and what do they inhibit?

Answer 13

Dickkopf, Frzb, and Tiki which inhibit Wnt signalling and Chordin and Noggin which inhibits BMPs

IGF is also secreted which inhibits both Wnt and BMPs

Question 14

What molecules are secreted from the notochord mesoderm and what do they inhibit?

Answer 14

Chordin, Noggin and Foliistatin which inhibits BMPs

Question 15

How does the inhibition of Wnt and BMP signalling compare between eary/mid stage gastrula and late stage gastrula?

Answer 15

• Early/mid stage gastrula inhibits both Wnt and BMP signalling
• Last stage gastrula only inhibits BMP signalling

Question 16

What is the consequence of the early gastrula organiser secreting BMP and Wnt antagonists?

Answer 16

Prevents BMP4 and Wnt8 signalling on the dorsal side and consequently head and brain tissue are induced

Question 17

What is the consequence of the late gastrula organiser only secreting BMP agonists (not Wnt)?

Answer 17

Only BMP signalling is inhibited which leads to the inductions of the trunk and spinal cord

Question 18

What is a default of embryonic ectoderm and what induces epidermal fate?

Answer 18

Neural fate is the default of embryonic ectoderm and BMP is an inducer of epidermal fate

Question 19

How do Noggin, Chordin and Cerberus (BMP antagonists) act on ectoderm?

Answer 19

Surpress epidermal fate and induce neural fate

Question 20

What does Cerberus, Frzb and Dickkopf (Wnt antagonists) induce and promote?

Answer 20

Induce anterior character within the neural tissue and promote brain development by antagonizing the posteriorizing and ventralizing functions of Wnt8 in the mesoderm, preserving prechordal mesodermal identity

Question 21

Outline the 2 gradient models of embryonic axis formation which are generated by the organiser?

Answer 21

1. An A/P gradient of Wnt signalling activity in the developing neural plate, which is lowest in anterior neural plate where Wnt antagonism is highest (from pharyngeal endoderm and prechordal mesoderm)
2. A D/V gradient of BMP signalling activity, that is lowest in the neural plate and the dorsal axial mesoderm, where BMP antagonist activity is highest (from Noggin in prechordal mesoderm and notochord)

Question 22

What are the 2 separate functions of Wnt signalling in the Xenopus embryo?

Answer 22

1. Specification of the D/V axis in the early embryo before gastrulation
2. Patterning of the A/P axis in the embryo after gastrulation

Question 23

Explain the involvement of Wnts in the specification of the D/V axis in the early embryo before gastrulation

Answer 23

• Induction of Nieuwkoop centre
• Accumulation of active beta-catenin the nuclei of the Nieuwkoop centre cells enables transcription of Organiser-specific Wnt antagonist and BMP antagonists

Question 24

Explain the involvement of Wnts in the patterning of the A/P axis in the embryo after gastrulation

Answer 24

• Ventralisation and patriotization of the mesoderm
• Dkk1, Cerberus, Frzb (Wnt antagonists) produced by the early organizer confer anterior character to anterior structures by inhibiting Wnt activity
• Creates a gradient of Wnt activity across the A/P axis
• Wnt activity specifies posterior (and ventral) fates.

Question 25

What induces the formation of the brain and spinal cord?

Answer 25

• Dual inhibition of Wnt and BMP signalling promotes brain formation in the anterior neural plate
• BMP inhibition without Wnt inhibition promotes spinal cord formation

Question 26

How are the detailed elements of A/P pattern in the brain and spinal cord created?

Answer 26

1. In the early gastrula, prechordal mesoderm/pharyngeal endoderm first signal to the anterior neural plate
2. This then activates anterior transcription factors that specify brain tissue
3. Forming notochord then represses anterior transcription factors in the mid-gastrula and by the late gastrula stage has activated posterior transcription factors in the spinal cord

Question 27

Explain the activation stage of the activation-transformation model of neural tube A/P patterning

Answer 27

1. The Spemann-Mangold organiser in the early gastrula secretes Wnt antagonists and BMP antagonists
2. Neuroectoderm exposed to these organiser signals adopts forebrain fate in the neural plate

Question 28

Explain the transformation stage of the activation-transformation model of neural tube A/P patterning

Answer 28

1. From mid-gastrula stage onwards, secretion of Wnt antagonists by the Spermann-Mangold organiser is attenuated
2. Posterior neural plate is exposed to multiple gradients of posteriorizing factors e.g Wnt, RA and FGFs
3. Forebrain fate is suppressed and over time, increasingly posteriorized neural fates are induced by posteriorizing factors
4. Neural plate elongates and transitions to neural tube

Question 29

What does the activation-transformation model of neural tube A/P patterning involve?

Answer 29

Involves simultaneous integration of information for multiple signalling pathways (Wnts, BMPs, RA and FGFs)

Question 30

How is the neural tube regionalised A/P?

Answer 30

1. Achieved by two opposing gradients of antagonistic signals- anteriorizing and posteriorizing factors
2. These opposing gradients introduce complex patterns of differential gene expression across the A/P axis, allowing high resolution regionalization of the CNS
3. Transcription factors expressed in the neural tube interpret the posteriorizing and anteriorizing signal intensities by transcribing different Hox genes in different parts of the neural tube which leads to the regional specification

Question 31

Which factors are anteriorising and posteriorising in the A/P regionalisation of the neural tube?

Answer 31

Anteriorizing factors = Wnt and BMP antagonists

Posteriorizing factors = Wnt, FGFs and RA

Question 32

How is the pattern refined in the vertebrate spinal cord and column?

Answer 32

• The distinct domains of the vertebrate spinal cord and vertebral column are defined through their specific combinations of Hox transcription factor expression
• RA gradient induces different patterns of Hox gene transcription at different positions along the A/P axis

Question 33

How is the pattern refined in the brain?

Answer 33

• In the brain, subdivision into forebrain, midbrain and hindbrain is regulated by posteriorizing factors such as Wnts, RA and FGFs
• An interaction between forebrain and hindbrain cells induces midbrain cells at the boundary between them
• Further events result in regionalisation of forebrain into diencephalon and telencephalon

Question 34

What is DLHP and what is its function

Answer 34

A group of cells which define the single cell thick epithelial sheet which induces the neural plate

Cells will delaminate from the neuroectoderm as neural crest cells, separating the close neural tube from the overlying epidermis

Question 35

What signalling systems polarise the neural tube along its D/V axis?

Answer 35

TGF- β ligands (mainly BMPs), Shh and Wnts

Question 36

How do BMPs, Shh and Wnt polarise the neural tube along its D/V axis?

Answer 36

1. TGF- β ligands (mainly BMPs) secreted by the dorsal roof plate induce dorsal neuronal fates. Initially, only BMP4 is expressed and it is localised to the roof plate but soon several TGF- β ligands are expressed in partly overlapping domains which extend from the roof plate to regions of the neural tube beneath
2. Shh secreted by ventral floor plate (and notochord) induces ventral neuronal fates
3. Wnt signals are also secreted by the roof plate and functions include promoting expression of BMPs in roof plate and contributing to induction of the neural crest

Question 37

Explain how different fates are induced by BMPs, Shh and Wnts

Answer 37

• In the dorsal neural tube, different members of the TGF- β family are expressed in different domains and induce different dorsal neuronal fates
• In the ventral neural tube, different concentrations of Shh induce different ventral neuronal fates
• A gradient of Shh protein is established in the ventral neural tube which determines neural progenitor fate by regulating expression of homeodomain transcription factors such as Pax7, Pax6 and Nkx6.1 in a Shh concentration dependant manner
• The neuronal subtypes specified at different positions along the D/V axis activates specific transcription factors in response to the particular combinations of TGF- β ligands or the concentration of Shh detected

Question 38

Outline the role of TGF- β and Shh together in the early neural tube

Answer 38

1. TGF- β and Shh induces distinct domains of transcription factor expression in neural progenitor domains along the D/V axis of the early neural tube
2. These transcription factors define distinct neural identifies that are realised when progenitors terminally differentiate into specific neurons a few days later

Question 39

Explain which different neuronal subtypes are induced by different concentrations of Shh

Answer 39

1. High Shh in the most ventral part of the neural tube induces Nkx6.1, slightly lower concentrations induce Pax7
2. Some neural progenitors express both Nkx6.1 and Pax6, whereas other progenitors express both Pax6 and Pax7

o Nkx6.1 only induces V3 interneurons
o Nkx6.1 + Pax6 induces motor neurons and V2 interneurons
o Pax6 only induces V1 interneurons