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Advanced topics in developmental biology > Lecture 5 - FGF and mesodermal gene regulation > Flashcards

Lecture 5 - FGF and mesodermal gene regulation Flashcards

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1
Q

Ouline Amaya

  • Aim
  • Progression of experiments
  • Findings
A

Aim

Show a role for FGFs in mesoderm formation

Progression of experiments

  1. Inhibited FGF signalling with dnFGFR
    • posterior truncation
  2. Three dn-FGFR constructs
    1. XFD: muscle, somites, notochord

Early events in development require FGF signalling for mesoderm formation

  1. Use XFD FGFR to block FGF signalling
    * Lose expression of brachyury (staining)
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2
Q

Outline Shulte-Merker and Smith

  • Aim
  • Progression of experiments
  • Findings
A

Aim

Show that FGFs are regulators of Brachyury expression

Progression of experiments (EF1a loading control)

  1. 5 Background observations
  2. The ability of brachyury with noggin to induce muscle gene expression is dependent on FGF signalling (RNAase, dnFGFR XFD)
  3. Brachyury activates FGF and brachyury (No tail, eFGF)
  4. FGF activates brachyury (Cbra, eFGF)
  5. FGF maintains brachyury (Dissociate gastrula mesoderm - inhibit FGF, look for brachyury, rescue with eFGF)
  6. FGF maintains activin nodal induced brachyury expression (time course, 90, 135, 200, 260. W/ FGF maintained (T), without FGF pulse (T) then lost)
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3
Q

Outline Branney

  • Aim
  • Progression of experiments
  • Findings
A

Aim

Identify othe targets of FGF signalling in the mesoderm with microarrays

Progression of experiments

  1. Microarray of 14000 genes in WT and FGF inhibited embryos
  2. Validation of samples
    1. Insitu of phospho ERK in WT/FGF inhibited
    2. Expression of cdx4, MyoD and Bra lost
  3. Scatterplot Microarray analysis of 14000 genes
    1. DN-FGFR1 v. control
    2. DN-FGFR4 v. control (above - FGF normally inhibit, below - FGF normally activate)
  4. Validate results
    1. Are known targets detected (Bra, Cdx4) and decreased
    2. Are the putative targets expressed in right place
      1. phospho ERK and FGF in mesoderm, Eph TKR and MAPK phosphotase in same region
  5. Built list of targets
    1. 2 fold change in expression, P
  6. DUSP5, MKP1, MKP3 downregulated when FGF blocked
    1. In situ around mesoderm when FGF present
    2. Western blot of animal caps without/with FGF, FGF induces DUSP5, MKP1
  7. DUSP proteins inhibit FGF signalling
    1. Animal cap explants: FGF4/FGF4+DUSP5/MKP1/3
    2. Western blot of levels of phospho ERK only present with FGF4 not FGF+MKP1/3/DUSP5
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4
Q

Outline the features of the fibroblast growth factor family structure

A
  • small polypeptide growth factors
  • 22 FGFs identified in higher vertebrates
  • 140 aa core with a conserved structure - important to binding to the receptors and interaction with glycosylated proteins
  • secreted (have typical amino acid terminal signal peptide for targette4d secretion through the periplam in the golgi) and non-secreted (presented on the outside of cells) forms
  • bind to heparan sulphate protoglycans (HSPG) in the extracellular environment
  • 20-30kDa
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5
Q

Outline the receptors involved in FGF signalling

A
  • Four high affinity receptors (FGFR1, 2, 3, 4)
  • Three extracellular Ig loops
  • single pass transmembrane domain
  • intracellular tyrosine kinase domain important for signalling
  • multiple isoforms from alternate splicing of the extracellular Ig loops
  • ligands, receptors and HSPGs interact to form the activated receptor complex
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6
Q

What is the process of FGF signal transduction?

A
  1. In the extracellular space, the FGF ligand binds and activates the FGF receptor -> activated FGF receptor complex (dimerises in the presence of 2 FGF ligands bound in a tripartite structure with HSPG)
  2. This initiates an intracellular signalling cascade. Activating either the Ras, PLC or PI3 pathways.
  3. The PLC pathways leads to the formation of diaglycerol and the release of calcium ions. The PIC kinase pathway leads to the activation of PKC.
  4. The activation of Ras activates the MAP kinase cascade, involveing 3 kinases (Raf (MAPKKK), MEK (MAPKK) and ERK (MAPK))
  5. In the nucleus activated MAP kinase phosphorylates target TF leading to the altered transcription of multiple target genes

This pathway can be regulated at multiple different levels by inhibitors (sprouty, MPK3)

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7
Q

How was it known that FGFs can induce mesoderm formation?

A
  • all the cells of the animal hemisphere can respond to mesoderm inducing siganls (Animal cap experiment for mesoderm inducing signals)
  • cultured animal caps in simple salt solution containing purified growth factors
  • TGFbeta family members activin and nodal are active in this assay (induce ventral mesoderm)
  • FGF family members active in this assay (induce ventral mesoderm)
  • instead of producing ball of epidermis

Therefore expected FGFs to be expressed in the ventral hemisphere as that is where TGFbeta family members expressed to pattern the mesoderm

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8
Q

How was it shown that FGFs are not expressed in the vegetal hemisphere?

A

FGF3, FGF4 and FGF8 are expressed and are active in the early mesoderm durning gastrula stages. Expected to be in vegetal cells as this is the source of primary mesoderm inducing signals TGFbeta family)

FGF4 mRNA in situ hybridisation: xpressed within the mesoderm at the start of gastrulation

FGF activity shown as antibody staining to phosphorylated ERK MAP kinase (as this is phosphorylated and activated by FGF signalling)

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9
Q

What was the aim of the Amaya study and how did they go about it?

A

Aim: demonstrate a role for FGFs in mesoderm formation

How: Inhibition of FGF signalling by the micro injection of mRNA coding for a dominant negative version of the FGFR1 (lacks intracellular typrosine kinase domain) into frog embryos, so that signal transduction is inhibited even in the binding of FGF to the receptor

Result: inhibition of FGF signalling gives rise to a dramatic phenotype of posterior truncation in xenopus 3 day embryo

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10
Q

How did Amaya show that FGF signalling is required for mesoderm formation?

A

Used 3 FGF-receptor constructs:

  1. d50 FGFR mutant that doesn’t function as a dominant negative
  2. HAVO FGFR mutant that doesn’t function as a dominant negative
  3. XFD FGFR mutant that functions as a dominant negative

Injected embryos

  1. doesn’t affect muscle development
  2. doesn’t affect muscle development
  3. inhibits muscle development

Seen though looking at molecular markers. Visualise mesodermal skeletal muscle derived from the somites by staining with a skeletal muscle marker - in XFD, this is dramatically reduced.

When stained with a notochord (most dorsal mesodermal structure) antibody, see that injection of XFD mRNA inhibits notochord development

Affects late events in development - differentiation of mesodermal structures

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11
Q

What are the features of the brachyury gene?

A

Brachyury:

  • Highly conserved gene important in the formation of mesoderm
  • gene originally identified via the T mutant in mice that has abnormal mesoderm development
  • T-box transcription factor co-expressed with FGFs in the early mesoderm of all vertebrates
  • required for all mesoderm development in vertebrates
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12
Q

How did Amaya show that FGF signalling is required for Brachyury expression in the mesoderm (early event in development - shortly after mesoderm induction)?

A
  1. Brachyury normally expressed in amphibian development in equitorial region as a response to the primary mesoderm signal
  2. if block FGF lose expression of Brachyury (early mesodermal marker)
  3. shown by in situ brachyury staining using FGFR dominant negative receptor mRNA injected into embryo
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13
Q

What was the aim and background observation of the Shulte-Merker and Smith study?

A

Aim: Demonstrate that FGFs are regulators of Brachyury expression

Background observations:

  • Brachyury is required for mesoderm formation in vertebrates
  • Brachyury induces mesoderm in animal cap explants
  • Activin/Nodal and FGF induce mesoderm in animal cap explants
  • Activin/NOdak and FGF induce the expression of Brachyury in animal cap explants
  • Brachyury is a transcription factor that regulates gene expression
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14
Q

What is the purpose of RNAase protection?

A

To semi quantitatively analyse gene expression

Band of the gel correponds with the intensity of expression

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15
Q

How did Shulte-Merker and Smith demonstrate that FGF is required for brachyury activity?

A

Use RNAase protection assay with the lanes corresponding to Animal cap explants

  • Noggin: secreted BMP antagonist important for neural induction
  • EFIalpha as loading control - shows not evenly loaded, varying in intensity
  • Brachyury in combination with noggin induces muscle gene expression (easliest isoform of muscle actin is cardiac muscle isoform)
  • inhibition of FGF (by dominant - FGFR) blocks muscle gene activation by brachyury
  • FGF signalling is required for the ability of brachyury to activate muscle gene expression

Hypothesis: Brachyury activates expression of FGF to initiate the induction of the mesoderm

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16
Q

How did Sulte-Merker and smith show that brachyury activates FGF transcription?

A
  • RNA protection analysis of eFGF from animal cap explants (embryonic FGF - amphibian ortholog of mammalian FGF4) in the early mesoderm
  • In the brachyury mutant there is no level above the uninjected control (no expression of eFGF)
  • But No-tail (fish ortholog of brachyury) is able to induce expression of FGF

Shows that brachyury able to induce expression of FGF ligands

17
Q

How did Sulte-Merker and Smith show that FGF signalling activates Brachyury transcription?

A
  • Looked at the expression of brachyury (Xbra) in various animal cap experiments analysed by RNAse protection
  • Animal caps treated with eFGF or bFGF - Showed Xbra expression
  • Treated with No-tail - got brachyury expression (able to induce own expression)
  • Used fish Brachyury (No-tail) so that can distingush between the induced endogenous amphibian brachyury expressed

The regulatory relatioships and co-expression of genes in teh mesoderm suggests that FGF and brachyury are components of a postivie feedback or autocatalytic loop

18
Q

Why might FGF and Brachyury be involved in a postive feedback loop?

A

Important to elevate the expression of both types of molecules in the early mesoderm

Brachyury necessary for the mesoderm formation of all vertebrates

19
Q

When it was found that FGF and brachyury may be involved in a postitive feedback loop whathypothesis was tested and how?

A

H: In the early mesoderm FGF signalling is required to maintain Brachyury expression

Experiment to test:

  1. Isolate pieces of mesoderm from gastrula stage embryos (ventral or dorsal)
  2. Dissociate cells in order to interfere with cell-to-cell signalling (e.g. FGF signalling)
  3. Analyse for brachyury gene expression
  4. Use exogenous FGF (bFGF) to rescue brachyury gene expression
  5. Used EFI-alpha as loading control

Results

  • In ventral and dorsal dissociated cells lose expression of brachyury
  • In associated cells have expression of brachyury (Xbra)
  • can be rescued by adding exogenous brachyury (bFGF) to the dissociated cells
20
Q

How was it demonstrated that FGF maintains Activin/nodal induced brachyury expression?

A

Prior: Prev. shown that activin/nodal/vg1 signalling induces brachyury expression

Aim: How does TGF-b fit into FGF-brachyury pathway/how does FGF inhibition affect activin induced brachyury expression?

RNAse protection/Animal cap experiments at diff time points

  • time points: 90 mins, 135 mins, 200 mins, 260 mins
    • uninjected, unmanipulated animal caps
    • animal caps with activin
  • HAVO mutant: at 135 mins get robust brachyury expression with activin
  • the activation of brachyury expression by activin is inhibited if FGF signalling blocked by XFD at the later stages
  • however at the earlier stages there is a small output of brachyury in the presence of FGF expression by activin

Conclusion: When FGF signalling is functional, the pulse of brachyury bought about by activin then induces FGF expression kicking off the autoregulation of expression. Need FGF to maintain activin/nodal induced brachyury expression

21
Q

How was it shown that brachyury expression is localised with FGF activity? What is the model for the role of FGF signalling in maintaining brachyury expression in the mesoderm?

A

In situ hybridisation of brachyury and FGF activity in mesdoerm.

Indictates that vegetally localised TGFbeta family members (Vg1 and nodal) induce apulse of expression of brachyury in the mesoderm which in term activates FGF expression. Intracellular FGF signalling maintains and amplifies brachyury expression in the mesoderm.

22
Q

What is the purpose and process of a micro array?

A

Purpose: analysing gene expression in parallel

  1. Knowledge of genome sequence allows the design of gene specific DNA probes that recognise mrNA corresponding to every individual gene expressed in an organism
  2. probes can be spotted at high density onto an array chip
  3. isolate an RNA sample and label it
  4. hybridise to microarray
  5. intenisty of hybridisation to individual gene probe is quantified and indicates the level of expession for the genes on the microarray
23
Q

How were novel genes that are regulated by FGF signalling in the mesoderm identified?

A
  1. Compare gene expression in control embryos to FGF inhibited (injection of mRNA coding for dominant negiative FGFR) embryos [experiment carried out in triplicate to allow statistical analysis of the data]
  2. Do subsequent experiemtns to validate the targets
    • expression analysis
    • sequence analysis
  3. Do functional studies to see what roles these genes have in regulating function
    • overexpression
    • inhibition
24
Q

Before undertaking microarray analysis what is it important to do?

A

Important to validate the quality of the samples

e.g. is FGF signalling effectively blocked

25
Q

How were samples analysed before undertaking the microarray analysis?

A

Use in situ hybridisation and compare gene expression in WT embryos at the start of gastrulation (after FGF signalling occurs) and embryos where FGF signalling blocked (dn FGFR1, dnFGFR4)

Early gastrula stage embryostained with an antibody that recognises the activated/phosphorylated ERK show that dnFGFR block ERK phosphorylation

  • dnFGFRs block expression of known FGF targets (MyoD, Cdx4, Bra)
26
Q

How do you present the results from a microarray analysis?

A

Scatterplot analysis: plot log ratios of group averages for DN-FGFR1,4 against the control

If there is no change in expression plotted points lie along the midline

If above midline; indicates an elevated gene expression when FGF signalling is blocked (therefore in normal development FGF would be inhibiting these genes)

If below midline; downregulated when FGF blocked (in normal development genes require FGF signalling for expression

Found that FGFR1 similar expression pattern to FGFR4; but FGFR4 gave a stronger response

27
Q

Following the presentation of the results from the micro array, how can you validate these results?

A

Look at data and establish whether known targets of FGF are detyected in the screen and expression affected as expected.

Found that:

  • Brachyury: expression reduced when FGF blocked as expected (FGF maintains brachyury expression)

Other known genes that expressed same pattern: Cdx4, M.coil, Iro3, Sprouty2

28
Q

How can novel genes thought to be involved in FGF signalling be validated post micro array analysis?

A

Determine whether these putative targets are:

  • expressed at the right time
  • expressed in the right place

Know where FGF expressed in the early embryo (by the spacoal distibution of phospho-ERK in the equatorial region)

Genes identified in the microarray analysis were consistent with the FGF signalling location

e.g. Eph tyrosine kinase receptor in equitarial region, MAP kinase phosphotase - all in a ring around the equator of the embryo

29
Q

In the microarray analysis, what change in expression level was used to determine a change indicative of significance?

A
  1. over 2-fold change in expression levels in response to FGF inhibition (hopefully avoid noise in data)
  2. P<0.01 using statistical analysis

List of putitative gnes:

  • 67 + regulated with FGGF signalling
  • 17 negatively regulated by FGF signalling

Then investigated the putaticve function of genes, many encode for transcription or signalling

30
Q

Give an example of a novel FGF related gene identified through microarray analysis

A

Dual specificity phosphotase proteins (DUSP)

31
Q

What are the features of dual specificity phosphatase (DUSP) protens? How are their expressions avtivated?

A
  • enzymes which dephosphorylate threonine and tyrosines in MAP kinase ERK
  • modulate the activity of the MAP kinase pathway
  • MKP1, MKP3 and DUSP5 are down regulated when FGF signalling is blocked (shown in in situ of DUSP5 expression when FGF inhibited, lose expression in equitoral region)
  • FGF induces DUSP5 expression: low level in animal caps, FGF can induce high expression when added of both DUSP5 and MKP1
32
Q

How was it shown that DUSP proteins inhibit FGF signalling?

A

Animal cap experiments

  • Control: forms cilliated epidermis
  • FGF4 added: fluid filled epidermis filled with mesodermal compnents
  • FGF4 + (MKP1/MKP3/DUSP5): lose expression of fluid filled vesicles, look like control

DUSP proteins inhibit FGF mesoderm induction.

Western blot of levels of phosphorylated ERK

  • GAPDH as loading control - same level for all
  • dp-ERK only expressed in FGF4 added to the animal cap, not for control or FGF4 + (MKP1/MKP3/DUSP5)

DUSP proteins inhibit FGF induced ERK phosphorylation.

33
Q

How are the DUSP proteins thought to act in FGF signalling?

A
  1. FGF signalling
  2. Leads to ERK activation
  3. Expression of target genes including DUSP proteins
  4. DUSP proteins (MAP kinase phosphotase) inhibit ERK activation

Negative feedback loop

34
Q

Following the identification of the DUSP proteins from the microarray analysis - what is the altered pathway for mesoderm formation?

A
  1. TGFbeta (Vg1) and Nodal induce a transient pulse of Brachyury expression
  2. FGF signalling elevates the levels of brachury
  3. positive feedback loop kept in check as FGF also activates inhibitory genes , e.g. DUSP
  4. This balance maintains gene expression at the appropraite lebel in the discrete region of the mesoderm

Advanced topics in developmental biology (10 decks)

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