Lecture 4 - The rise, fall and rise again of a very interesting molecule Flashcards
Describe the prevailing model for amphibian mesoderm induction
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Describe the post translational processing of TGF-b related proteins
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Discuss the importance of maternal contribution to early amphibian development
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What experiments demonstrated that Vg1 is a localised maternal product
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What experiments investigated the processing and biological activity of Vg1?
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What experiments demonstrated that Vg1 is required for normal mammalian development?
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What is a triploblast?
Having three primary germ layers
What are the three primary germ layers of early vertebrate embryos?
- The ectoderm - forms the epidermis and central nervous system
- The mesoderm - forms the notochord, dermis, skeleton, muscle, kidney, heart and blood
- The endoderm - forms the gut, liver, pancreas and lungs
What does the ectoderm form?
The ectoderm - forms the epidermis and central nervous system
What does the mesoderm form?
The mesoderm - forms the notochord, dermis, skeleton, muscle, kidney, heart and blood
What does the endoderm form?
The endoderm - forms the gut, liver, pancreas and lungs
How are different types of mesoderm induced?
Different types of mesoderm are induced by TGF-b-like proteins (block the formation of the mesoderm) acting as morphogens
There is a nodal gradient from the endoderm (vegetal hemisphere) acting on the equitorial region. High concetration to low concentration dorsal to ventral.
What does TGF-b pattern the mesoderm in which way?
High dose: Dorsal medorm to form the notochord
Intermediate dose: At the lateral mesoderm to form the skeletal muscle, heart and kidney
Low dose: at the ventral mesoderm to form the smooth muscle and blood
What are the two types of TGF-b signalling?
- Activin/Nodal: Mesoderm induction
- BMP signalling
What is the general process of TGF-beta signalling
TGF-betas are dimeric ligands which bind to two receptors (receptor type II - Ser/Thr kinase domain and receptor type I - GS box) leading to SMAD activation by phosphylation (Nodal like ligands - SMAD 2,3; BMP-like ligands - SMAD 1,5) when these transcription factors are activated they activate SMAD 4 to regulate gene transcription or repression
What SMAD TFs does signalling by nodal-like ligands activate?
Nodal like ligands: SMAD 2,3
What SMAD TFs does signalling by BMP like ligands activate?
Signalling by BMP ligands: SMAD 1, 5
What is mauration of TGF-beta peptides?
post translational processing at the ER and golgi
targeted to the ER and the golgi by an amino terminal signal peptide (20-30 aa long)
biologically activates TGFbeta dimer
What is the process of maturation of the TGFbeta peptides?
- Translated as large pre-pro-peptides
- In the ER and golgi dimers are formed and the pre-pro region is removed
- A dimer of the mature peptide (7 conserved cysteines form disulphide bridges of 2 monomoers) is secreted and biologically active
Why do maternally depositied mRNAs and proteins play a critical role in early amphibian development?
No rtanscription of the zygotic genome until cell division 12; vegetal cells are able to signal before the onset of zygotic transcription
When does zygotic transcription begin in amphibian development?
Zyogtic transcription begins at the mid blastula transition (MBT)
When are nodals expressed?
Nodals are expressed zygotically
Why are early signals in the amphibian embryo though to be maternally deposited?
- Mesoderm induction begins before the onset of transcription from the zygotic genome (begins at the mid-blastula transition (cell division 12))
- Nodals are expressed zygotically
- vegetal cells are able to signal before the onset of zygotic transcription
How can the expression of nodal in theamphibian zygote be visualised?
In situ hybrisation of TGFbeta Nodal
see grsadient of colouration dorsal to ventral
TGFbeta signalling requires TGFbeta signalling but these have not yet been expressed therefore there must be a member of the TGFbeta deposited in the early embryo
What was the purpose of the Rebagliati study and what was known prior to the conduction of the experiemtn? How?
Aim: identify localised maternal mRNAs
Knew: Cells can signal to induce mesoderm
Hypothesis: Factors responsible for pre-MBT sigalling must be localised to the vegetal signalling
How: Differential screen to identify factors present in different regions of the embryo involved in the process
How are phage cDNA libraries generated?
Phage cDNA libraries are a way of propagating individual cDNAs
- Present all cDNAs cloned in library to an E.coli lawn
- Clear spots are bacteria infected with an individual phage clone representing a single propaged cDNA
- Bacteria lyse and phage DNA exposed to environment
- lay nylon membrane on the surface, DNA adhears to the membrane corresponding to the individual clones
- DNA from the plasmid and phage vectors can be transferred and immobilised to nylon membranes
- Hybridise radioactive antisense probes to the membranes
- Transfer can be done multiple times
How is a differential screen done once the cDNA library is generated? (Identification of localised maternal mRNAs)
- cDNa phage library constructed from RNA present in the egg
- Isolate animal and vegetal localised mRNAs
- Generate radiolabelled animal and vegetal probes
- plate out egg cDNA library and make replica filters
- screen cDNA library with animal and vegetal probes (hybridise probes to filters) on the replica filters
- Isolate and sequence differentially expressed clones
- Found Vg1 vegetally localised
What are the features of Vg1 identified by Weeks and Melton?
- Vg1 cDNA: 2.3 kb
- Vg1 protein: 41.8kd
- contains an amino terminal signal peptide
- 7 cystein residues highly conserved in TFG-betas
- cysteines are required for the secondary structure and dimerisation
- typical TGFbeta structure
What presented TGF beta as a potential candidate for mesoderm induction?
- localised expression in the vegetal hemisphere
- TGFbeta structure homolgy (tGF beta family)
- expressed at a particular point in development
Aside from the differential screen, how did Rebagliati prove that Vg1 mrNA is localised to the vegetal hemisphere?
Used northern blot of disected pieces from animal and vegetal hemisphere
- Isolated RNA from the A/V regions of the egg in the embryo
- Ran on gel
- Transfer to membrane
- Label with probes from vegetal cDNA
- found high levels of hybridation in the vegetal region
(used histone for loading control)
Aside from the differential screen, how did Rebagliati prove that Vg1 mrNA is localised to the vegetal hemisphere at a particular point in development?
Northern blot of developmental time course in oocyte,blastula and gastrula stages
Found Vg1 present in maternal phase before the onset of zygotic transcription
(No loading control - some may have been missed out/ loaded with less sample
Aside from the differential screen, how did Rebagliati prove that Vg1 mrNA is localised to the vegetal hemisphere and expressed at the right time?
- Northern blot of dissected pieces
- Northern blot of developmental time course
Ideally, how would you analyse gene expression and protein localisation?
- Protein localisation over mRNA expression
- Ideally determine protein localisation with an antibody
- Carry out immuno-histochemistry on sections or whole pieces of tissue
- protein visualised by fluoresence or enzyme based colour reactions
- however it is difficult and expensive to make antibodies
What can be used instead of looking at protein localisation by antibody staining?
mRNA localisation by in situ hybridisation
- sensitive
- technique is applicable to any mRNA
What are the caveats of mRNA localisation by in situ hybridisation?
- mRNAs are post-transcriptionally regulated at multiple levels
- presence of mRNA does not guarantee presence of the protein
Outline an in situ hybridisation
- Generate antisense probes regonising target mRNA (radioactively label, label antisense RNA, chemically label)
- hybridise to target RNA in tissue
- wash extensively
- visualise RNA hybrids either:
- Radioactively labelled; use photographic emulsion and silver grains
- Chemical label; antibody and enzyme linked colour reaction
How did Weeks and melton (1987) and Thomas and Moos (2007) show maternal Vg1 mRNA is localised to the vegetal hemisphere?
Weeks and Melton 1987
Radioactive label - showed Vg1 mRNA in Xenopus oocyte, tight localisation to the vegetal cortex
Thomas and Moos 2007
Chemical label - Vg1 mRNA in a Xenopus blastula, widespread in vegetal hemisphere
But neither looking at protein
What criteria are necessary to fulfil to assign function to a molecule?
- relevent biological activity
- present at the right time
- present at the right place
- is it neccessary
How were the functional critera for Vg1 being an endogenous materal mesoderm induced fulfilled?
- relevant biological activity
- Dale (BMP-Vg1 mRNA induces mesoderm, animal cap explants)
- present at the right time
- Rebagliati 1985 (differential screen)
- Rebagliati 1985 (northern blot of developmental time course)
- present at the right place
- Rebagliati 1985 (differential screen)
- Rebagliati 1985 (northern blot of dissected pieces)
- mRNA Vg1 in situ hybridisation by Weeks and melton 1987 (radioactive label, xenopus oocyte, tight to vegetal cortex); Thomas and Moos 2007 (chemical label, xenopus blastula, widespread vegetal hemisphere)
- is it neccessary
What problems did Dale encounter working with Vg1?
Looking at biological activity
TGFbetas have complex post translational modification
Found that Vg1 is neither processed nor secreted in oocytes, it does not form dimers, synthetic Vg1 has little biological activity
How did Dale show that Vg1 is neither processed nor secreted in oocytes?
- Inject oocytes with synthtic Vg1 mRNA
- Label translation products with 35S-methionie
- Immunoprecipitate with anti-Vg1 antibody
- separate out on SDS-PAGE
- Autoradiography - get radioactive bands when exposed to x-ray film
- If secreted should appear in the cultre medium - doesn’t appea r in the culture medium. Found unprocessed pre-pro-protein. OT: trpsin to release proteins stuck to the surface. OF: removed follicle cells.
Control oocytes injected with egg white proteins from chick embryo which are produced and secreted in abundance
How did Dale show that Vg1 did not form dimers?
- Inject oocytes with synthetic Vg1 mRNA
- Label translation products with 35S-methionine
- Immunoprecipitate with anti-Vg1 antibody
- SDS-PAGE in reducing and non-reducing conditions (b-mercaptoethanol or DDT (reduces cystein bonds important for forming dimeric TGFbeta protein) alongside Ig heavy and light chain as control
- Autoradiography
- Little difference in non-reduced and reduced conditions - nothing corresponding to the size of the predicted mature dimeric protein
How did Dale demonstrate that synthetic Vg1 mRNA has little biological activity?
Lots of experiments with injecting synthetic Vg1 into embryos
- Injection of Vg1 mRNA does not affect normal development
- Vg1 might synergise with other mesoderm inducers - when combined Vg1 treatment with other mesoderm inducing factors identify an increase in mesoderm inducing activity in the presence of Vg1
- Lack of activity correlates with a lack of post-translational processing
Following the identification that synthetic Vg1 mRNA has little biological activity, how did Dale test their hypothesis tha tht eVg1 pre=pro region affects the processing of mature Vg1 proteins?
Generated a synthetic cDNA coding for a fusion protein containing Vg1 mature region and the preproregion of BMP4
How did Dale demonstrate that BMP Vg1 is processed and forms fimers in oocytes?
Did a time course with oocytes injected mRNA coding for fusion Vg1
- Inject oocytes with synthetic BMP-Vg1 mRNA
- label translation products with 35S-methionine
- Immunoprecipitate with anti-Vg1 antibody
- SDS-PAGE in reducing and non-reducing conditions
- Autoradiography
Found: most protein consisted of unprocessed pre-pro-protein under the reduced and non-reduced conditions but also see lower molecular band of the predicted processed monomeric protein in reduced conditions and the predicted mature dimeric protein in the non reduced conditions
It is processed!
How did Dale show that the BMP4-Vg1 C-terminal region is secreted by oocytes?
Figure 6
M (1), MS (2), MH (3): medium surrounding oocytes injected with Vg1 or BVg1
O (4), OS (5), OH (6): oocyte extract injected with Vg1 or BVg1
S and H potential the secretion of mature protein
Under reducing conditions:
- WT Vg1 - no appearance of a band indicating processing and secretion
- BVg1 - see appearance of band in the media correspoding to the unprocess and processed monomeric protein
How did Dale show BMP4-Vg1 is biologically active? (induce mesoderm)
Animal Cap assay
- Take animal cap explant (cells that give rise to the ectoderm but are able to respond to mesoderm inducing signals - if culture in medium give rise to ectoderm, can stain for markers of the epidermis e.g. keratin)
- Animal cap cells exposed to mesodermal inducing factors transform to fluid filled vesicles containing mesodermal factors e.g. skeletal muscle, blood
- Take animal cap from embyro and inject with BMP4-Vg1
- Produce fluid filled vesicles
Vg1: ball of cilliated epidermis
BMP-Vg1: hollow vesicles containing skeletal muscle, blood
Therefore the mature Vg1 protein has mesoderm inducing biiological activity
How was it shown that Vg1 is essential for development?
Birsoy et al 2006
As maternal deposited factors can’t use eggs deposited by the mother as factors already present.
- Remove oocytes from ovary (amenable for microinjection)
- Deplete Vg1 from oocytes by injecting oocytes with inhibitory reagents
- Mature oocytes in vitro
- Stain oocytes with vital dye
- put back into mother frog
- eggs are laid and fertilised
- identify depleted embryos for analysis
What two types of inhibitory reagents might be used to deplete Vg1 in embryos?
- Antisense morpholino oligos
- blocks translation of target mRNAs by base pairing to initiating methionine
- Antisense phosphorothioate oligos
- depletes target mRNAs as one of the O atoms in the backbone is replaced with a S atom, binds to mRNA and targets for degradation
What controls were used by Birsoy et al to check inhibitory reagents were working?
Real-time PCR
- increased concentration of phosphorothoate antisense oligos, showed it resulted in a reduced levels of Vg1
- levels of Vg1 depleted - Vg1 mRNA sent for degradation
Antibody for Bg1 on western blot
- alpha tubulin as loading control
- increasing amounts of antisense MO at different stages of development to show a depletion of the Vg1 protein
- show maternal depletion and translation blocking
How did Birsoy show that Vg1 affectsdevelopment?
Used Vg1 depleted embryos
Showed that Vg1 depleted embryos have reduced heads and abnormal gastrulation movements (mesoderm drives gastrulation)
Looked at the effect of Vg1 depleteion on a range of markers known to be involved in mesoderm induction:
- chordin: BMP inhibitor/antagonist involved in neural induction ,secreted by spemanns organiser
- Cerbeous: BMP antagonist
With increasing amounts of inhibitory reagents, the expression of organiser markers (chordin, cerbeous) is reduced in dorsal meosderm
Western blot of phosphorylated SMAD
TGFbeta signalling causes phosphorylated SMAD 2,3 (required formesoderm induction)
Would expect a depletion of phosphorylated SMAD 2,3 with more inhibtory reagents of Vg1
At highest levels of injection of phosphorothiolate oligios, get reduced levels of pSMAD 2 and increased levels of pSMAD1 furing late blastula stage
How did Birsoy demonsntrate Vg1 induction in vivo?
Looked at the expresion of genes brachury, chordin and FGF8 as markers of mesoderm
- Compare expression in whole embryo to cap alone, cap on WT base and cap on VG1- base
- Isoolate animal cap, hardly any expression
- Animal cap on vegetal tissue have high levels of brachury, chordin and FGF8
- WT animal cap on Vg1- base, highly reduced levels of expression of these genes
Vg1 depleted vegetal cells therefore have a reduced ability to induce mesoderm
Why was it so hard to prove Vg1 had biological activity?
- Xenopus laevis is pseudo-tetraploid
- Has two forms of Vg1: Vg1(S) and Vg1(P)
- polymorphisms between alleles of the two genomes; pseudo allele has a different protein coding information for Vg1 - differ by the presence of a proline/serine
- Protein in other frog genomes has a serine at this particular position in the secretory signal peptide
- Substitution of P means protein only poorly processed
- The original Vg1 clone studied is the Vg1(P) form which is not processed
What data was used to back up the Vg1(S) vs. Vg1(P) hypothesis of processing of the Vg1 gene?
Western blot of oocytes injected with Vg1(S/P)
Most of the protein at both oocyte and stage 10 us the 41kDa unprocessed size
There is some processing of Vg1(S) in oocytes and embryos; additional smaller band
But this doesn’t line up with the 14kDa of the mature protein
There is extra processing of the serine version but don’t know what
What ecperiment did Birsoy do to deonstrate the Vg1(S) has biological activty?
- Vg1(S) used to rescue the Vg1 depleted phenotype
- Depletion phenotype assumes the specific depletion of a single molecule
- To prove the phenotype is specifically arising due to the targeted depleted molecule must rescue with this single molecule and get WT phenotype
- Vg1 -: gaping blastopore
- Vg1 - Vg1(S) + : reduces closure of the blastopore
- Repeated with the head/tail embryo - see partial rescued phenotype
-
western blot
* deplete Vg1 there a depletion in pSMAD2 (but not clear), restored when resuced, alpha-tubulin as control - show rescued expression of mesoderm markers with Vg1(S) previously shown to be inhibited by Vg1 depletion
What is the status of maternal factors pre-MBT?
Vg1 gradient from the vegetal hemisphere acting on the equitoral region
High concentration at dorsal, low condetration at ventral
Initiates mesoderm induction in the equitorial region
What is the status of zygotic factors post-MBT?
Nodal gradient acting in a dorsal (high conc) to ventral (low conc) manner released from the vegetal hemisphere acting to pattern the mesoderm
