Lecture 6 - FGF and neural induction

Question 1

What is the role of BMP inhibition on the induction of anterior nerual tissue?

Answer 1

1.

Question 2

Describe the basic anteroposterior strcuture of the early nervous system

Answer 2

*

Question 3

Describe the IGF anf FGF signal transduction pathways

Answer 3

1.

Question 4

Discuss the experiments indicating a role for FGF in neural induction

Answer 4

1.

Question 5

DiCuss the experiments indicating that SMAD1 is an interaction node for the BMP and MAP kinase signal transduction pathways

Answer 5

1.

Question 6

What is the involvememt of BMP4 antagonism in the gastrula embryo

Answer 6

• BMP4 potentiates ventral fates in the mesoderm and ectoderm
• BMP inhibiition important for neural indcution in all animals
• all deutrosome and peutrosomes use BMP signalling and antagonists to pattern the dorsal mesoderm
• Spemanns organism is the dorsal most source of BMP antagonists (Noggin, Chordin) inhibit BMPs
• Activity of BMP4 in the ventral ectoderm allows differentiation of the epidermis
• zone of no BMP activity in the ectoderm important in neural induction

Question 7

What happens to the position of the three germ layers during gastrulation?

Answer 7

• Gastrulation starts with the formation of the dorsal blastopore lip
• Cells of the mesoderm are internalised
• dorsal mesoderm comes to underlie the dorsal ectoderm
• signalling process of BMP antagonism continues as dorsal mesoderm underlies the dorsal ectoderm

Question 8

What occurs in neuralation?

Answer 8

BMP antagonists give rise to region void of BMP (during neurulation) - in anterior region of the neurula

Neural plate rolls up

Question 9

What is the mode of action of Noggin and Chordin?

Answer 9

• Noggin, chordin and other neural inducers (follistatin) are secreted molecules
• do not bind to cell surface receptors; bind to and inhibit the function of bone morphogenic proteins (BMP - members of the TGFbeta family)
• BMP proteins would otherwise bind to cell surface receptor and initate a signal transduction pathway to activate genes neccessary for the formation of the epidermis e.g. keratin

Question 10

What is the anteroposterior pattern of the nervous system?

Answer 10

Nervous sytem consists of regionalised neural tisse

3 sibdivisions

• Forebrain (prosencephalon)
• Midbrain (Mesencephalon)
• Hindbrain (Rhonbencephalon)

If inhibit BMP signalling get formation of the forebrain, not the others

Question 11

What moecules induce anterior neural tissue?

Answer 11

BMP4 antagonists (chordin and noggin) induce the expression of neural genes expressed in the forebrain and midbrain

do NOT induce the expression of neur\al genes ecpressed in the hind brain and s[inal cord

Use molecular markers:

• Otx2: important for the function of various tissues in the forebrain, homeodomain TF
• En-2: expressed at the junction between the midbrain and the hindbrain
• Krox20: 2 stripes in the hind brain
• HoxB9: expressed in the most posterior part of the neural tissue

Noggin and chordin only able to induce expression of Otx2, not more posterior markers

Question 12

What work did Lamb and Harland’s Nerual induction and patterning by FGF study build upon?

Answer 12

• Previous study suggested FGF can induce neural tissue in gastrula stage animal cap cells
• Various technical issues indicated this needed further investigation

Original Assay

1. took animal cap explant and dissociated them into single cell ‘soup’
2. Treated with FGF
3. FGF found to potentiate the formation of neural tissue

Critisms

• to mimic neural tissue induction requires BMP activity in the ectoderm - does this technique not inhibiting BMP signal, biasing the cells to form neural tissue by inhibing BMP signalling

Question 13

What questions did lamb and harland seek to answer?

Answer 13

1. Will FGF induce neural tissu in intact ectoderm
2. Does FGF induce neural tissue in the absence of mesoderm

Question 14

Why did Lamb and Harland think FGF was a good canditate for posterior neural induction?

Answer 14

• Location of expression in normal development: FGFs are expressed in the dorsal mesoderm derived from the organiser, already known to be a source of neural inducing signals
• Shown by an in situ with ab staining for FGF3, FGF4 and FGF8: expressed and active (ab staining for phosphorylated ERK MAP kinase) in the early mesoderm during gastrula stages
• FGF4 continues to be expressed in the notocord

Question 15

What is the animal cap assay? How was this used to test whether FGF induces neural tissue?

Answer 15

1. Blastula stage animal cap compenent to respond to mesoderm inducing signals
2. Take animal cap explant at late blastula stage (to avoid interferance from underlying mesoderm - at the gsatrula stage mesoderm begins to underlie the ectoderm potentially exposing to an inducing signal)
3. culture to gastrula stage equivelent in low calcium and magnesium containing medium (Why: animal cap has two layers, outermost and inner layer. Only the inner layer is able to respond to mesoderm and neural inducing signals. Tends to ‘heal up’ when cultured in normal mesoderm so inner layer completely surrounded by outer layer and inner layer can’t be exposed)
4. treat explants with FGF and FGF in combination with BMP antagonists
5. assay for gene expression of molecular markers (neural markers and regional subdivision of neural tissue)

Question 16

What is a double in situ hybridisation?

Answer 16

Allows the analysis of the expression of 2 genes simaltaenously by visualising their expression with different colours

Question 17

What were the results of the FGF/BMP antagonist animal cap experiments by Lamb and Harland and how were these visualised?

Answer 17

Visualised by a double in situ hybridisation

Looked at (NRP-1 and M.actin) or (Otx2 and En2) or HoxB9 in normal embryo, untreated animal cap cells, and animal cap cells treated with FGF

• NRP-1: Neural marker
• M.actin: mesodermal marker
• Otx2: anterior neural marker
• En2: Interior neural marker
• HoxB9: posterior neural marker

1. Can FGF induce neural tissue in the absence of the mesoderm?When stained with NRP-1 and M. actin, FGF induces some animal caps to express M. actin, some also express NRP1, some express both. Some cells expressing NRP1 in the ansence of mesoderm - neural marker in the absence of mesoderm = FGF can induce neural tissue in the absence of mesoderm
2. Doesn’t induce expression of the most anterior tissue (Otx2, En2), different from the type of tissue you get from BMP antagonist
3. High levels of expression of posterior tissue (HoxB9) - FGF induces posterior character tissue

Question 18

How did Lam and Harland demonstrate the interaction of BMP antagonists with FGF signalling?

Answer 18

• Stained control embryos, animal cap explants w/ noggin or FGF or Noggin and FGF, with NRP-1 and M.actin
• Noggin alone: NRP1 expression
• FGF alone: micture of NRP1 and M. actin expression (some neural, some mesodermal )
• Noggin and FGF: inhibits residual mesoderm induction, get the robust induction of NRP1 neural marker, get elongated polarised structures

FGF and noggin syngergise in neural induction, but get polarised structures with posterior neural tissue and anterior neural tissue - WHAT IS IN THE MIDDLE

Question 19

How was the interaction of Noggin and FGF to induce Neural tissue investigated further?

Answer 19

• Inhibited BMP with a constant level of noggin and titrated in concentrqations of FGF to determine whether FGF acting in a morphogen dependent manner.
• Used double in situ hybridisation of: Otx2 (most anterior neural) and HoxB9 (most posterior nerual)
• With an increasing concentration of FGF, get more noggin and FGF and polarised structures
• Have anterior markers at one end and posterior markers at the other end, with no overlap in some structures
• Needed to identify teh regional tissue in the middle of the xplant

Question 20

How was the tissue in the centre of the anterior and posterior polerised structure generated by the addition of Noggin and FGF to animal cap explants?

Answer 20

Used same technique, and stained instead with Krox20 and M.actin in double in situ hybrisation.

Frox20: expressed in two striped in the hind brain, more posterior than Otx2 but more anterior than HoxB9

When animal caps were treated with noggin: no expression of Krox20

When treated with FGF: some expression of Krox 20, some explants with robust expression

When treated with noggin and FGF: Expressed in a stripe in the midle of the tissue

Shows able to generate whole patterning of CNS by FGF and BMP antagonism in combination (synergistic interaction)

Question 21

Aside from noggin and FGFs working in synergy, what other signals are involved in patterning the neural tissue?

Answer 21

Retinoic acid, WNT signalling

Question 22

What is competence?

Answer 22

The ability of a cell to respond to a particular signal

Question 23

How does the competence to FGF signals change during development?

Answer 23

Competence of animal hemisphere cells changes from blastula to gastrula stage

• blastula stage cells respond to FGF by forming mesoderm
• gastrula stage cells respond to FGF by forming neural tissue

Basis of this change could e changes in the epigenetics of target genes

Question 24

What are the similarities of IGFs and FGFs?

Answer 24

• Insulin-like growth factors (IGFs) and fibroblast growth factors (FGFs) both signal through dimeric tyrosine kinase receptors (RTKs) and activate the MAP kinase pathway
• FGFs signal through homo- or heterodimers of FGFR1-4 held together by disulphide bonds
• IGfs signal through a tetrameric receptor consisting of 2 alpha subunits and 2 beta subunits held together by disulphide bonds. Signal by binding to the alpha subunit and causing dimerisation
• Both have intracellular tyrosine kinase domains and transmembrane domains

Question 25

Where are IGFs expressed?

Answer 25

In situ hybridisation: In the early embryo, in the animal hemisphere and maternally, then in the dorsal midline (where neural induction occurs). IGF2, 3 and R in the dorsal midline.

• Xenopus IGfs and IGFR were cloned
• Dominant negative form of IGFR made
• DN-IGFR consists opf a secreted extracellular domain

IGF overexpression: If inject mRNA encoding for mRNAs, translation, and overproduction of cement glands (normallyy v small in normal development at the anterior end, often closely associated with the neural tube)

IGF inhibition: by overexpression of the dominant negative form of IGFR. Block IGF signalling and lose induction of the neural structures marked by loss of Otx2 (anterior neural marker) and eye

Conclude: IGF also involved in neural induction

Question 26

How did Pera et al integrate the IGF, FGF and anti-BMP signals?

Answer 26

In situ hybridisation with 2 markers

• Sox 2: marker of the neural plate
• N-tubulin: marker of differentiated neurons in the neural plate
• Sox2 expression in control embryo at early neurula stage confined to the neural plate, N-tubulin in the control embryo expressed in those cells beginning to differentiate as neurons in the neural plate
• under increaased expression of chordin, IGF, FGF the expression of Sox2 and N-tubulin expands.
• IGF, FGF and chordin increase he size of th neural plate

Animal cap experiments

Treatments: WE, AC control, chordin, chordin and controlMO, Chrd and IGFR-MO, Chd and DN-IGFR-4a, chd and BMP7

Markers: NCAM (general neural), Rx2a (anterior neural), a-actin (mesoderm), EF1alpha (control)

Results:

WE: NCAM, Rx2a (no mesoderm)

AC control: nothing

Chd: NCAM, Rx2a (+ controlMO)

Chd + (IGFR-MO/DNFGFR-4a/BMP7): nothing

Need IGF or FGF to induce neural tissue by chordin

BMP7 also reduces ability of chd ot induce neural tissue

Question 27

What is the process of BMP signalling?

Answer 27

• signal through cell surface serine/threonine kinase receptors
• activate intracellular SMAD proteins which are TF and activate gene expression of target genes necessary to form epidermis
• BMP singals through SMAD1,5, which then may activate SMAD 4

Question 28

What is the background to the hypothesis that SMAD1 is a node for interaction between the BMP, IFG and FGF signalling pathiways? And that the basis of IGF and FGF neural induction is MAP kinase inhibition of SMAD1 activity?

Answer 28

1. Smad1 is a transcriptional effector of BMP signalling
2. BMP signalling phosphorylates Smad1 and promotes nuclear translocation
3. IGF and FGF signal through the MAP kinase pathway
4. Smad is phosphorylated by MAP kinase
5. The MAP kinase phosporylation of Smad1 inhibits nuclear translocation

Question 29

What are the hypothesise for the integration of the IGF, FGF and anti-BMP signals through Smad1?

Answer 29

1. Smad1 is a node for interaction between the BMP FGF and IGF signalling pathways
2. The basis of IGF and FGF neural induction is MAP kinase inhibition of Smad1 activity

Question 30

What is the effect of phosphorylation of Smad1 by BMPsignalling FGF signalling?

Answer 30

BMP signalling

• phosphorylates the C terminus of the Smad1 protein to target Smad 1 to the nucleus

FGF/IGF signalling

• phosphorylates a group of cysteine residues in the linker region which counteracts the effect of the phosphorylation of the C terminal

Question 31

What different ways are there of inhibiting BMP signalling?

Answer 31

1. Secreted BMP antagonists inhibit activity by binding BMP
2. MAP kinase signalling by FGFs and IGFs by phosphorylating the linker region inhibits the ability to translocate Smad1 to the nucleus

Question 32

How was Smad1 as an interaction node for BMP/FGF/IGF signalling investigated?

Answer 32

Created a series of Smad1 mutants

• WT-Smad1
• LM-Smad1 (linker serine residues phosphorylated by the MAPK converted to alanine residues which cannot be phosphorylated - LM cannot be inhbited (super SMAD) but can be activated)
• DM-Smad1 (both linker serine residues and c terminal s residues converted to alanine (and v) cannot be phosphorylated at all)
• CM-Smad1 (Cterminal serine residues converted to alanine residues, cannot be phosphorylated by the BMPR)

Smad1 proteins were epitope tagged to allow the monitoring of protein levels - all found to be at similar levels

Results

Used markers (Sizzled - a ventral non-neural marker induced by BMP signalling; N-tubulin - a dorsal neural marker induced by BMP inhibition) to determine the functional consequences of LM-Smad1 mutation

• If LM-Smad acting as a super-SMAD would expect a potentiation of Ventral gene expression over dorsal gene expression - extra neural type tissue
• WT-Smad: potentiates formation of ventral type tissue higher expression of sizzled than control but not of N-tubulin
• LM-Smad: dramatic change in the appearance of the embryo, lose dorsal axis, upregulation of ventral marker at the expense of the dorsal marker

Question 33

What was the purpose of epitope tagging Smad1 proteins?

Answer 33

To rule out the potential that these synthetic RNAs are not translated at the same level, therefore accounting for differences in biological activity

Using epitope tagged allows investigate protein levels in a quantifiable manner, or for subcellular localisation when there is no antibody available

• using recombinant DNA technology, make fusion protein to a sequence of amino acids that have a good antibody for
• e.g. FLAG tag
• quantify by antibody recognising epitope

Found that all SMADs have similar levels of translation

Question 34

How do the Smad1 mutants affect neural induction in vivo?

Answer 34

Markers

Sox2: Neural plate

N-tubulin: differentiating neurons

Results

LM-SMAD: decrease in sox2 expresion and loss of N-tubulin expression - dramatic inhibition of neural induction

Question 35

How do the Smad1 mutants affect neural induction in vitro? (Explant assay)

Answer 35

Animal cap assay

Treated AC explants with Chd, FGF8 or IGF2 in the presence or absence of WT-Smad1 and LM-Smad1

Results

LM-Smad1 able to block the ability of FGF8 and IGF2 to induce neural tissue because it cannot be phosphorylated by MAPK downstream and the basis of NI by FGF and IGF is the phosphorylation of serine residues in the linker region of Smad1

Critisms: doesn’t demonstrate these serine residues are phosphorylated

Question 36

How was it shown that the Smad1 linker region is phosphorylated in vivo?

Answer 36

Western blot - whole embryos

3 markers:

• ERK2, phosphorylated ERK, Smad1 and pSmad1

Levels of protein analysed at 3 stages:

• 8 (blastula stage), 10.5 and 12.5 (gastrula stage - when FGF signalling definitely occuring and active in the mesoderm and overlying ectoderm)

Results

• no changes in the overall levels of ERK
• when FGF active, get upregulation of pERK demonstrating FGF active at these stages
• as there was no antibody for phosphorylated Smad1, in order to see if this lead to changes in the phosphorylation state of Smad1, had to rely on changes in the mobility of Smad1 protein when phosphorylated
• WT: when FGF signalling active get appearance of higher molecular weight band (representing changes in mobility of Smad1 as a result of phosphorylation)
• LM-Smad1: No changes in the mobility of the protein therefore phosphorylation of Smad1 lost even though FGF active
• Linker regions of Smad1 neccessary for FGF to inhibit Smad1 activity by phosphorylation during gastrulation

Critisms:Not looking at the endogenous protein, does the phosphorylation at the C terminus not affect mobility. Not direct demonstration that this affects phosphorylation at the linker region

Question 37

What is the model for the integration of BMP inhibition and MAPK signalling during neural induction?

Answer 37

1. BMP signalling leads to the phosphorylation and activation of Smad1 at the C-terminal region
2. This inhibits neural induction and promotes the formation of the epidermis in the ventral ectoderm by activation of relevent genes
3. Smad1 need to be inhibited to form neural tissue by Noggin and Chordin binding BMP or by MAP kinase signalling inhibiting nuclear translocation (activted by IGF, FGF)
4. The combination of these two inhibitory functions (and more) are important for the formation of the neural tissue

Results in ectoderm patterning:

• high BMP and low MAP kinase = epidermis
• low BMP and high MAP kinase = neural

Question 38

How is FGF signalling involved in posterior neural development

Answer 38

• Inhibition of Smad1 signalling by chordin and noggin only gives anterior neural development (FGF/IGF also probably contributes)
• FGF also leads to the activation of more posterior neural markers in a parallel pathway
• These in combination lead to a fully patterened neural vertebrate system