BMS237 Advanced Developmental Biology Flashcards
Why is developmental biology research important in todays society?
Because todays society is ageing faster than it is growing
- By 2020 - the population will grow by 3% and the population over 65 will grow by 12%
How much of the UK welfare budget is spent on pensioners?
55% - £114bn
What are the two theories of how organisms develop?
Epigenesis
- Aristotle 324BC
- Organisms develop progressively through the generation of new structures and forms
Preformationism
- Organsims develop from miniature versions of themselves: homunculus
What is the cell theory?
- Proposed by Robert Cooke, 1665
- That all organisms are composed of one or more cells
- The cell is the most basic unit of structure, function and organisation in all organisms
- All cells arise from pre existing living cells
How was the cell theory discovered?
Reported his studies - viewed thin slices of corks and looked into microscopes and saw cells
What is ‘Germ plasm determinants’ theory?
Weismann in 1880s
- Germ cells have a collection of ‘determinants’
- Somatic cells only have some of these determinants and therefore will carry out a specific function based on the determinants present
What experiments were conducted to try and prove Weismanns theory?
William Roux - 1888
- Killed one cell in a two cell stage frog embryo and left it in contact
- Half of the embryo developed suggesting that that cell only had those determinants (he was wrong)
Hans Driesch
- He physically separated the cells at two and four stage
- All developed normally and fully
- Showing that all determinants were present in every cell
What was the main difference between Roux’s and Driesch’s experiment?
In Driesch
- The two cells didn’t remain in contact so lost cell to cell communication - causing them to develop into full organisms
What are the two types of cell division?
Symmetric and asymmetric
- asymmetric produces two distinct daughter cells
What are the types of cell - cell communication involved in developmental biology?
Paracrine
- Signal produced by one cell and acts on a different cell
Autocine
- Produced by one cell and acts on the same cell
Juxtacrine
- Cell to cell contact
What is required for cell to cell interaction?
Signal reception requires cells to be competent (permissive
environment, receptor and transduction components)
What are the common features in signal transduction pathways?
- Reception: The ligand (growth factor, signaling molecule) binds to a cell surface receptor and activates it.
- Transduction: Receptor activation induces the transduction of the signal from the membrane to the nucleus via a cascade of secondary messenger activation.
- Response: A transcription factor is activated and induces the transcription of specific target genes.
What are the three germ layers and what do they develop into?
Ectoderm - Nervous system, skin
Endoderm - Gut
Mesoderm - Blood, heart, muscle, kidney
How is tissue homeostasis controlled in development?
Negative feedback loop
- Controls the amount of growth that occurs
- Produces a protein which acts on stem cells preventing further proliferation
What is the role of stem cells in adults?
Stem cell mediated repair
What can happen when tissue homeostasis is disrupted?
If cells are pushed more towards differentiation instead of stem cells then it leads to ageing and degeneration
If cells are pushed towards progenitors and stem cells then leads to cancer
What are the processes who underlie embryonic development?
Pattern formation
Morphogenesis
Cell differentiation
Growth
What is pattern formation?
The process by which cells are organized in space and
time to produce a well-ordered structure within the embryo
- Development of body axis
Why is the development of body axis important?
Allows cell to know exact coordinates and allow it to adjust its genetic program and acquire new characteristics
What is morphogenesis?
Cell and tissue movement, and changes in cell behaviour
that give the developing organ its shape in 3D
eg. Gastrulation
What processes contribute to morphogenesis?
Cell adhesion
Cell migration
Cell death
Cell shape
What is cell differentiation?
Process by which cells become different from each other and acquire
specialized properties. Governed by changes in gene expression, which
dictate the repertoire of protein synthesised
- Gradual acquisition of new characteristics and loss of pluripotency
What are the steps involved in cell differentiation?
Stem cell Specification Determination Differentiation Maturation
What is the difference between specification and determination ?
Specification
- Early commitment (unstable)
- If taken out of environment and placed in another it would not keep the same lineage and develop a new fate
Determination
- Commitment becomes stable
- Would not change fate if implanted in a new environment
What is growth in development?
Continuous process (embryonic, fetal, post-natal, adult). Growth rate varies depending on age and organ. Cell proliferation, cell enlargement, accretion.
What is accretion?
Accumulation of ECM around the cell in order to grow
Is differentiation reversible?
Yes
- Add specific transcription factors and it returns to a pluripotent form
- Induced pluripotent stem cells
When are animal models useful for developmental research?
Funnel model - Wrong
- That they are only useful in very early development
- Decreases as development progresses
Hourglass model - Right
- Early stages can vary a lot between animals
- The mid embryonic stages are the most useful as they share a lot of similarities with humans
What experimental approaches are used in developmental biology to study when and where a gene is expressed?
- in situ hybridization,
- northern blot,
- RT-PCR,
- micro-array
- reporter lines (transgenic)
What is in situ hybridisation used for?
Study when and where gene expression occurs in an embryo
Outline the method for in situ hybridisation
Generate a probe (nucleotide sequence) that mimics the gene of interest and label it
The probe allows us to detect this compound using immunohistochemistry
- Using an antibody that is coupled with an enzyme (alkaline phophostase)
- Generates a chromogenic reaction where hybridisation of a probe to a specific transcript occurs
Hybridisation is very stables so creates a striped pattern which can be seen in the developing embryo
Why is in situ hybridisation qualitative and not quantitative?
Because the chromogenic reaction will continue to get stronger as the reaction is left
- There is no correlation between amount of transcript present and the intensity of colour
What are reporter lines used to study in developmental biology?
Where and when a gene is expressed in a embryo
Outline the method for reporter lines
Information about regulatory sequences (promotors and enhancers) are required
- If you have information regarding this then you use that molecular information to drive expression of another gene
- Use green fluorescence protein - drive its expression
- Fluorescence is observed
Is the information received for reporter lines qualitative or quantitive?
Qualitative
What is a genome wide study?
Global information of overall gene expression of a specific cell
Give some examples of genome wide techniques used in developmental biology?
RT PCR
Microarrays
RNA seq
What are microarrays used for?
Finding gene expression content
Outline the technique for microarrays
- Reverse transcriptase to produce cDNA
- Label each cells cDNA with different fluorescent tags e.g.. cell A will all be red and B green
- Hybridize on a life which have a collection of short sequences that are specific to every single gene in the genome of that species
- If cell A was expressing a specific gene then a hybrid would form and red fluorescence would be seen , if B then green
- Can use software that screens the dots and looks at intensity of fluorescence - tells us the amount of gene present
What happens if there is co expression of genes in microarrays?
If cells A and B express different genes then red and green would be expressed
- No co expression
If genes expressed in both then would see yellow
Why do developmental biologists also have to investigate protein distribution?
Because proteins may not be expressed where it was transcribed
In development there are mechanisms that may delay the protein production so that transcription occurs
How is protein distribution investigated it?
Western blot
Immunohistochemistry
Outline the technique of immunofluorescence
Requires the availability of antibody specific to the protein of interest
Antibody specifically binds protein
Detect this antibody by adding a second antibody specified for the immunoglobulin for the species that the original antibody is raised in
Compare western blotting and immunoflourescence
Western blot tells amount of protein in tissue but does not have as a high as a resolution
How would you study how important a protein is in development?
Gain of function and loss of function experiments
Define loss of function
Mutation in a gene that disrupts the expression or the function of the protein product encoded by the mutated gene
Define gain of function
Mutation in a gene that confers a gain in the activity of
the protein product encoded by the mutated gene.
Define forward genetics
Seeks to identify a gene whose mutation caused a
particular phenotype
Define reverse genetics
Seeks to characterize the phenotype of particular
mutated gene
What are the two ways of causing loss of function mutations?
Forward genetics
- Introducing random mutations in genomes and look for phenotype
Reverse genetics
- Disrupt function of specific genes
What animal models can have forward genetics done?
C elegans
Drosophila
Zebrafish
Mice
What is used to introduce a mutation in forward genetics?
ENU - N-ethyl N-nitrosourea
What are the disadvantages of forward genetics?
Time consuming - so usually done on animal models with short generation time
Expensive
What animal model can knockouts occur in?
Mice, drosophila and zebrafish
What is required for a knockout to occur?
Knowledge about the gene you’re knocking out
- Its structure and location on chromosome
Outline the formation of a conventional knockout mouse
- Create a construct that is similar to the endogenous gene (structure)
- Modify this construct with a gene (e.g. antibiotic resistant) that will disrupt function of endogenous gene - e.g. effecting start codon stopping transcription
- Once made the construct introduce into embryonic stem cells from a mice line (of one coat colour)
- Can select out embryonic stem cells that have incorporated your gene by growing the stem cells on antibiotics - only ones with the construct will survive
- Triggers homologous recombination - in cell division cross over reactions occur in chromatids causing replacement of endogenous gene
- Inject these cells into blastocyst and then surrogate mother
- Breed mouse and eventually will have a conventional knockout mice where the construct is in every single cell of the mouse
How can you check if the embryonic stem cells containing the construct are present in the knockout mouse?
By looking at the level of chimerism in coat colour as ES stem cells and blastocyst where from mice with different coat colours
What is the problem with conventional knockout mice?
The gene will have been knockout out from the earliest stage of development meaning that if that gene was required for normal embryonic development, it would be unlikely that the knockout mice would survive long enough to study the protein of interests function
How is the problem with conventional knockout mice overcome?
Conditional knockout mice crossed with tissue - specific cre mouse
Outline how a knockout mice can be made where the gene is only knocked out in specific tissues
- Construct will contain targeting gene but will be surrounded by loxp sequences which does not disrupt endogenous gene function
- Homologous recombination requires another mouse which expresses tissue specific cre recombinase - an enzyme that recognises loxp sites and triggers homologous recombination specifically in those tissues. The cre recombinase must be under the control of a promotor and enhancer
- Cross the conditional mouse wth the tissue specific cre mouse then knockout will occur later in development
How can you chose when a knockout of a gene occurs?
Variants of cre mice that are inducible that allows you to not only choose which tissue but also when the knockout occurs
How do you investigate when a gene is regulated?
- Embryology: tissue manipulation (graft, ablation)
- Manipulating signaling pathways: drugs, transfection/electroporation, genetics
Give examples of how tissue manipulation have lead to important embryological discoveries
Grafting different regions of developing embryo to different places and looked at the effect - lead to discovery of the organiser
Similar experiment lead discovery of the zone of polarising activity in the limb bud
How are the origins of cells expressing gene of interest investigated?
Fate mapping
- Label small group of cells in early development before cells have become committed
- Use antibody to detect the labelling later in development to see what has happened to the cell
What animals can fate maps be done in?
Zebrafish
Xenopus
C.elegans
How do you label cells in fate maps?
Injecting fluorescence dye
Adding a reporter gene
How can you label individual cells in a developing embryo?
Using loxp and cre recombinase
What is a morphogen?
A soluble secreted molecule that acts at a distance to specify the dates of cells. It specifies more than one cell type by forming a concentration gradient
Who came up with the idea of morphogens?
Alan Turing 1951
What is required of a morphogen make to make a concentration gradient?
Its not just making the morphogen that is important but also destruction of the morphogen at the other end of the field of cells
How can bilateral symmetry form?
If morphogens from both ends and sink in the middle
What must a morphogen do?
- Induce different outputs at different concentrations
- Act directly at a distance
How can you use the morphogens property of inducing different outputs at different concentrations to test if a molecule is actually a morphogen?
Morphogens are instructive not just permissive
- ectopically implant the morphogen and if its is a real morphogen it will change the cell fate if not the the cell will goto original fate (permissive)
- Can also make concentration of the morphogen the same in all of the cells then all cells would have the same fate
What happens if you ectopically implant Shh in the anterior of a chick limb bud?
Get duplication of the digits
- Shh is acting as a morphogen
How can you use the morphogens property of acting directly at a distance to test if a molecule is actually a morphogen?
The morphogen must be able to diffuse all of the cells to reach the other end
Signals that are morphogens do not diffuse through they trigger a new molecule to be produced at each cell and released
- genetically engineer to make the morphogen juxtacrine - stopping it passing through the membrane of neighbouring cells
- If its a morphogen then cells won’t change fate
- If not then still will change fate
What is meant by a signalling molecule being ‘bucket brigade’?
The molecule doesn’t diffuse trough its fuel of cells it binds to the membrane of the neighbouring cell and triggers a new molecule to be formed and released from that cell
- This new molecule then triggers the same response
How would making a genetic mosaic test if a molecule is actually a morphogen?
Remove the receptor for the morphogen on later cells I the field
- If actual morphogen cell fates won’t change in response to morphogen because won’t be able to detect it
- If bucket brigade then it won’t be affected and cell fates will still be induced as long as the directly neighbouring cell still has a receptor for it
How does diffusion establish a morphogen concentration?
Binding to molecules in the extracellular matrix (e.g. heparan sulphate proteoglycans) and high concentrations of receptor can generate a steep gradient - called “restricted diffusion”
Rapid degradation of the signal in the extracellular space may also generate a steep gradient
How do Heparan sulphate proteoglycans (HSPGs) regulate morphogen diffusion?
HSPGs are found in the extracellular matrix and are known to bind to many ligands. They are sometimes called co-receptors. They regulate morphogen diffusion by:
- sequestration or slowing diffusion. eg BMP (TGFbeta)
- facilitating diffusion (Hedgehog)
What is planar transcytosis?
When the molecule inputs from one side of the cell and outputs from the other side - doesn’t leave the vesicle
What is the role of planar transcytosis in morphogen gradients?
A pit forms in the cell membrane and engulfs the morphogen in a vesicle
Repeated cycles of endocytosis and resecretion allows certain morphogens to travel through the cells in a tissue
Give evidence for the role of planar transcytosis in establsihing morphogen gradients
Evidence for transcytosis in Dpp (TGFbeta) signalling: antibody staining shows that Dpp is found in vesicles AND mutations that block vesicle formation cause Dpp to act in a juxtacrine manner
How does timing affect morphogen concentrations?
All neighbouring cells will think thy are going to be a cell that requires little or no morphogen until the morphogen gradient is established and they received the instructions for their real fate
- There is a checkpoint at which all cells differentiate but not understood why
- As the gradient is established, gene expression is changing with time. There must be a mechanism to block premature specification. The cell probably waits for the steady state of receptor activation to be achieved - but the molecular mechanism for this “check point” is poorly understood.
What is the transcriptional read out model?
How varying morphogen concentrations lead to different cell fates
- Higher concentration of morphogen often results in a higher concentration of an activated transcription factor
- In this model, receptor activation causes transcription factor to enter the nucleus and direct transcription. It is the same transcription factor in every cell - keep in mind these cells are initially identical.
- It is the amount of transcription factor present that decided the cells fate
Give a transcription factor that also acts as a morphogen
Bicoid
How are transcription factor concentration interpreted at a DNA level?
Binding sites for transcription factors that activated by high concentrations of morphogens have a lower affinity and those activated by low concentrations have higher affinity binding sites
- Even though both sets of genes see the same levels of transcription factors, only the high affinity enhancers can bind enough transcription factors to activate gene expression.
- Also presence of negative feedback - repressor that switches off low concentration activated genes when high concentration genes are activated
How are strict thresholds of morphogen concentrations achieved even when the gradient isn’t that steep?
Positive feed back may help the cell commit to its fate. For example if one of the blue gene encodes a transcription factor that among other things activates its own expression
Why is the drosophila useful to use as a model organism?
Because it allows us to see evolutionary conserved genes between them and humans
What temperatures are drosophila live stock kept at?
18 decrees C
- If want to grow slowly with little maintenance
25 degrees C
- If want to grow fast
How long is development of drosophila?
10 days at 25 degrees C
21 days at 18 degrees C
What is the life span of drosophila?
40 days
Outline the history of using drosophila as a model organism
1910 - Morgan discovers a white eyed fly; white (w)
1913 - Sturtevant constructs first genetic map, genes are arranged in a linear order
1914/16- Bridges shows that chromosomes must contain genes.
1927 - Muller shows that X-rays cause mutations & chromosomal rearrangements
1979/1980 - Christiane Nüsslein-Volhard and Eric Wischaus undertake a saturation mutagenesis to identify genes involved in the development and patterning of the larval cuticle
1980s,90s - technical and methodological advances
What technical and methodological advances were made from drosophila?
P-element transformation- transgenics Enhancer trap - promoter trapping Gal4/UAS - gene misexpression FLP/FRT - ‘clonal’ mutant analysis RNAi- both ex vivo and in vivo omic’ technologies - transcriptome, proteome etc
When was the genetic map of the drosophila published?
24th March 2000
How can we use the genetic map of drosophila to investigate genomes?
Can align genomes of different drosophila species and see which parts of the DNA have been conserved through time as the species have evolved over millions of years
Which parts of DNA have been seen to be conserved in drosophila?
Protein encoding exons are highly conserved
Most introns are not conserved
Some introns appear to be conserved through time, why is this?
Because they have regulatory roles
- May be binding sites that transcription factors bind to
- Enhancer regions
What are the physical differences between male and female drosophila?
Males are smaller with darker tip of abdomen
Females are larger and have distinct segments
Explain courtship in drosophila
It is genetically encoded, not learnt
Very strong drive for reproduction
Males face females and buzz wings and dance
Outline spermatogenesis in drosophila
Males have two testes and sperm tails
At the tip there are germ line stem cells which are maintained by hub cells
Hub cells produce a ligand that maintains stem cell fate of the cells surrounding them
When the stem cells differentiate, one cell remains by the hub cell and the other grates away to differentiate into sperm cells
How do hub cells maintain the fate of stem cells in drosophila?
They produce JAK/STAT pathway ligand
What are ovarioles?
String like structures which are present in drosophila ovaries
Over 100
They bind together and are where eggs develop and mature
How does fertilisation occur in drosophila?
After conception, females store the sperm in seminal receptacles for 2-3 weeks and is used to fertilise her eggs
How do oocytes form in drosophila?
Stem cell niche keeps stem cells in stem cell fate
They divide and one cell moves away
The cells undergo cystoblast mitotic divisions
Divide into 16 cells, two of the cells will have four connections and one of these cells will become the oocyte and the other 15 cells will becomes the nurse cells
Oocytes grow at the expense of the nurse cells
Egg activation occurs and undergoes meiosis
What are polytene chromosomes?
Large chromosomes in the drosophila
- Made of sub bands - over 5000 each 22kb
What are the maternal contributions to the zygote of drosophila?
pronucleus and proteins are RNA
- Proteins and RNA are made in nurse cells and are transferred to the developing oocyte via ring canals
What is responsible for the location of maternal factors in drosophila eggs?
Microtubules - minus and plus ended motors
What happens in early development of a drosophila zygote?
Pro nuclei from egg and sperm fuse
8 divisions of nuclei occur
After the 14th division there will be thousands of nuclei
Some remain in the centre to become the nuclei of yolk cells and other migrate to the top
Membrane starts to upgrow and form around the nuclei forming cells
What are the common intracellular signals in drosophila?
Hedgehog Wingless (Wnt) Delta Transforming growth factor Fibroblast growth factor
What processes are inched in the conversion of a single cell into an embryo?
Morphogenesis
Cell proliferation
Differentiation
Migration
What is cell differentiation?
Process by which cells become different from each other and acquire specialised properties
Governed by changes in gene expression, which dictate the repertoire of protein synthesised
Cells that become specialised lose potency
What is a morula?
64-128 uniform cells - this is due to cell proliferation and occurs very quickly and occurs very little growth
How is morula split into halves?
Split into a top and a bottom hemisphere In xenopus - top hemisphere is animal - bottom is vegetal In chicks/humans - top is epiblast - bottom is hypoblast
What differences are seen in the oocyte and why is that important in embryo development?
At the 1 cell stage, before fertilisation, distribution of cytoplasmic components isn’t even
The oocyte is polarised and some of its components are only found in the vegetal half
When division occurs (in the animal vegetal plane) the cells become fundamentally different to each other as the top dividing cells will not inherit vegetal cytoplasmic components but bottom cells will
What is the first plane of division?
Animal vegetal
How does the presence of certain cytoplasmic components in different cells in a dividing morula cause a different fate?
The cytoplasmic components act on the DNA to regulate gene expression the vegetal hemisphere
- As a result particular transcriptional factors are turned on in the vegetal hemisphere and not in the animal
=> differences
How many genes does one transcription factor regulate?
Thousands
What transcription factor is turned on in the vegetal hemisphere of the morula?
VgT
What do changes in osmolarity in a morula lead to?
Blastocoel formation
What is the difference between the morula in a xenopus and a chick/human?
The morula is much flatter in chicks and humans and may be depicted as two flat surfaces
What does the activation of VgT transcription factor lead to?
Binds to the promotor region of Nodal gene up regulating it
What is the role of the nodal protein in the morula?
It is exported out of the cell in which it was made (made in vegetal cells) and is transported up into animal hemisphere
- Acts as a morphogen
- Animal hemisphere cells that recieve high concentrations of nodal become endoderm
- Lower levels of nodal become mesoderm
- No nodal protein become ectoderm
Which part of the morula of the germ layers develop from?
The animal hemisphere
The vegetal hemisphere are yolky energy providing cells which eventually die
How does the role of nodal differ in xenopus and in chick/humans?
Nodal also causes the cells to migrate and redistribute as well as just forming the germ layers
What would happen if the vegetal hemisphere didn’t exist in a morula?
All cells would have an ectoderm fate
How does a second symmetry event occur in the morula?
Sperm always binds to the animal hemisphere of the oocyte
- Opposite side to sperm binding activates Wnt signalling pathway which activating the transcription factor beta catenin
- So on the opposite side of the embryo Wnt signalling is activated leading to the formation of quadrants
- as horizontal axis from animal/vegetal and then vertical axis from beta catenin presence
What is meant by a morula having four quadrants?
The morula is split in half by animal/vegetal hemisphere
it os then split in half vertically as beta catenin is present only in the side of the morula that the sperm doesn’t bind - activating Wnt signalling
What is special about the morula quadrant which is on the vegetal side and has beta catenin?
That is the quadrant with the most nodal signalling and the most Wnt signalling
This nodal and beta catenin overlap is called the Nieuwkoop centre
The future dorsal and posterior side
Where does the organiser develop?
In the cells that lie just above the Neiukwoop centre as they receive the most nodal signalling acting a specialised sub set of genes
Why does the combination of high levels of nodal and beta catenin lead to the development of the organiser?
Two parts of the promotor for genes (such as Gsc) need to be activated in order to activate transcription
Therefore requires the binding of SMAD2/4 (a downstream effector of nodal) and beta catenin (a downstream effector of Wnt)
How can we tell that there are differences between the mesoderm and the organiser?
Through in situ hybridisation
- can see that different parts of mesoderm express different transcription factors and genes
- Eg. Brachyury is induced in response to low levels of nodal, Gsc and Chordin
- Xnot and Clim are induced in response to high levels of nodal and where Wnt signalling is activated
What happens to the organiser?
It undergoes convergent extension whereby the cells of the organiser form a long thin rod and internalise under the the ectoderm
Froms axial mesoderm
Why does the organiser undergo convergent extension?
Because the transcription factors expressed in the nucleus of cells of the organiser (e.g. siamois and goosecoid) begin to bind to promotors in an intrinsic manner to alter the cells behaviour
- including changes to migration and differentiation
What makes up the axial mesoderm?
Prechordal mesoderm and notochord
Why is there a clear AP axis after convergent extension of the organiser occurs?
Anterior end expresses Wnt and BMP antagonists
Posterior end expressed Wnt, FGF and retanoic acid
Antagonist actions from opposite ends creates a gradient forming the AP axis
What controls the formation of the AP axis?
Hox codes
How does neural induction occur?
Organiser produces secreted molecules - BMP antagonists such as Chordin and Noggin
These diffuse into the ectoderm and interfere with BMP interacting with receptor on a responding ectoderm cell
Causes the phosphorylation of SMAD
Causes a cascade of transcriptional factors to cause a neural fate
What happens in ectoderm cells that do not recieve BMP antagonists?
BMP is able to bind to BMP receptors on the ectodermal cells
Activating transcription factors that induce a skin fate
Where do BMP antagonists produced by the organiser diffuse into to?
Ectoderm
Mesoderm
Endoderm
What happens when BMP signalling is inhibited in the mesoderm?
Somites are formed
Give experimental evidence for neural induction
1920’s: Spemann and Mangold
- A donor organiser was grafted from a donor onto a host newt
- found that a ‘twinned’ embryo developed with a secondary neural axis
- Secondary neural tube was host derived showing it was induced from the ectoderm in response to signals from the organiser
- The prechordal mesoderm ad notochord where donor derived showing they’re formed from the organiser
Why was Wnt named that?
Because its an amalgam of wingless (the Drosophila gene) and int, a vertebrate proto-oncogene that encodes a mouse homologue of wingless.
When were the first wingless alleles first described?
The early 70’s
What is the relationship between Wg and Hh?
Wingless and Hedghog maintain each other so if you disrupt either of them it will lead to the same result
- Will cause wingless flies and disrupted segment polarity of the embryo
Why are there more types of Wnt in vertebrates then in drosophila?
Because vertebrates are more evolved so during evolution, duplications of the genes occurred
Its an ancient gene that is found in all metazoic organisms
How is the Wnt ligand formed?
When the ligand enters the secretory pathway, the N terminus is cleaved off
It becomes modified on two positions - (cys77 and ser209) by palmitoylation and palmitoleic acid modifications
Causes two fatty acids to be covalently linked to the wnt protein
How is Wnt secreted?
Wntless is required
- Needs to either form multimers or be loaded onto a lipoprotein
- Seen that HSPG is also important
Is it essential that Wnt is diffused out for normal function?
Evidence in drosophila say that just cell contact is enough for normal function of Wnt but no evidence to say this is the same in other organisms - most likely requires diffusion
What are the main receptors for Wnt?
Frizzled Arrow or (LRP in humans)
What is the main transcription factor activated from wnt signalling?
Beta catenin
Describe the Wnt receptors
Frizzled
- Made up of 7 transmembrane domains
- LRP stands for LDL receptor related protein
- LRP and Arrow are a single pass transmembrane protein which also makes contact with Wnt
How does Wnt bind to frizzled?
It binds to the long N terminal extension of frizzled which is known as the cysteine rich domain (CRD)
Give an example of an extracellular Wnt inhibitor?
Dickkopf1 (DKK1)
- Binds to LRP and Kremen promoting the internalisation of LRP
How is beta catenin broken down when Wnt signalling is not present?
There is destruction complex made up of APC, Axis, CK1, GSK3, Slimb proteins
Beta catenin binds to this complex and is phosphorylated CK1 and GSK3
This causes the Slimb protein to cause ubiquination of beta catenin leading to its degradation
What occurs to beta catenin when canonical Wnt signalling is present?
It is not broken down
- Wnt binds to Frizzled and Arrow causing them to be brought together leading to phosphorylation of Dsh
- This recruits the destruction complex to move to the receptor casino further phosphorylation of arrow creating binding sites for destruction causing the loss of Slimb from the destruction complex
- Beta catenin still binds to the complex and it is still phosphorylation but Slimb is not there to tag beta catenin with ubiquitin to target it for degradation
- This means that beta catenin remains in the complex causing it to clog
- Beta catenin can then leave the complex and enter the nucleus where it can activate transcription
How is beta catenin targeted for degradation by the destruction complex?
Phosphorylation by both CK1alpha and GSK3 (kinases) is required for beta catenin recognition by an E3 Ubiquitin ligase complex (which contains β-TrCP/Slimb) to cause subsequent degradation by the proteosome.
Explain the binding sites for β-TrCP/Slimb on beta catenin?
CKI phosphorylates a site in the N-terminal tail of beta catenin at a specific recognition site
Only after this occurs can GSK3 phosphorylate another recognition site. (GSK3 phosphorylates 3 times at 2 different sites)
β-TrCP/Slimb can then bind to beta catenin once phosphorylation of the 3rd recognition site by GSK3 has occurred
Describe β-TrCP/Slimb and its interactions
β-TrCP/Slimb is the interacting site of an SCF E3 ubiquitin ligase complex
- Contains a Roc component which binds to E2
- It contains an F box which is responsible for its interactions with Skp1
- It interacts with the substrate by a domain made up of WD40 repeats
What occurs in the nucleus when beta catenin enters?
When beta catenin is present
- It binds to the promotor and activates acetyl transferases and BRC1 ( a chromatin remodelling enzyme)
- Also activates transcription factors Pygo and Lgs
- Allows transcription to occur
What is the role of Groucho in the nucleus?
When beta catenin is not present
- Groucho represses transcription by recruiting histone deacetylases wjocj causes acetyl groups to remove from histones causing tighter packing so it is less accessible for transcription
What examples of negative feedback are there in the Wnt signalling pathways?
DKK1, DKK4 and Axin 2 are all produced during the Wnt signalling pathway but act negatively on the action of the pathway
What other pathways are Wnt signalling involved in?
Planar cell polarity and convergent extension
- Involved Wnt11, Wnt3, Frizzled, Rho, Roc, Vang11/2
Axon guidance
- Repulsion by RYK receptor tyrosine kinase
What Wnt can inhibit canonical Wnt signalling?
Wnt5a
- Via ror2
What is the role of planar cell polarity?
To make all cells point in one direction
eg. hairs on rats, bristles in flies legs, hair cells in ear
What occurs in a mutant of Vang12?
Convergent extension does not work successfully
- Vang12 plays a role in the Wnt signalling pathway
What is the role of canonical Wnt signalling in drosophila?
Segmentation, also expressed at D/V boundary of the wing required for patterning and outgrowth
- The first wingless gene
What is the role of canonical Wnt signalling in C.elegans?
Doesn’t have a vital role but is used to regulate neuronal fate
- If Wnt5 is active then the QL.d neurone will move backwards instead of forwards
- Useful investigating the roles of Wnts and the strength of the signals
What is the role of canonical Wnt signalling in zebrafish?
Wnt11 induces a dorsal fate due to its activation of beta catenin
Wnt8/3 induces ventral/posterior fates in the late zygotic stage
Loss of Axin1 due to Wnt activation leads to posteriorisation of the anterior brain causing the loss of eyes
What is the role of Wnt signalling in intestinal stem cells?
Required to maintain intestinal stem cells in stem cell fate
- If there is a loss of TCF4 or an over expression of dkk4 (and therefore loss of Wnt signalling) then you can see that there is a loss of these stem cells
- Can be investigated using in situ hybridisation
How can Wnt signalling lead to cancer?
Ectopic wnt signalling can occur by the loss of APC gene (in destruction complex)
If the other copy of APC gene on the other chromosome is lost to (eg. due to mutation) then it can cause familial adenomatous polyposis as stem cells will just continue to divide uncontrollably without differentiation - cancer
What types of cancer can faults in Wnt signalling lead to?
Familial adenomatous polyposis Hepatocellular carcinoma Breast cancer Ovarian and uterine cancers Melanomas Prostate cancer
What other diseases can faults in Wnt signalling lead to?
Wnt3-tetra-amelia
- Loss of function in Wnt3
- Babies born without limbs
Bone diseases
- LRP5 point munition cause insensitivity to Dkk causing an increase in bone density
- Axin2 mutations can cause severe tooth agenesis (oligodontia) - multiple missing teeth
What did Christiane Nüsslein-Volhard and Eric Wischaus do in 1979?
Undertook a saturation mutagenesis to identify genes involved in the development and patterning of the larval cuticle
What did Christiane Nüsslein-Volhard and Eric Wischaus find from their experiment?
They found 580 genes that didn’t cause lethality
Put them into complementation groups to check if they were on the same chromosome
Found that there were in 139 different genes in this experiment
How did Christiane Nüsslein-Volhard and Eric Wischaus check if the genes were on the same chromosome?
They crossed each gene with each of the other genes
If a phenotype was shown then it failed to complement, if no phenotype was shown then complemented
What mutations did Christiane Nüsslein-Volhard and Eric Wischaus find?
Lots including
Knirps - causes a loss middle chunk of segments
Paired - only has alternate segments
Gooseberry - lose half of each segment - segment polarity gene
What are the stages that occur during development that lead to segmentation?
Maternal genes
Gap genes
Pair rule genes
Segment polarity genes
What is bicoid?
A DNA binding transcriptional activator that acts as a morphogen
It is maternally loaded into the developing oocyte (by nurse cells)
What occurs if there is a loss of bicoid?
Loss of anterior structures
How can you partially rescue a bicoid mutant oocyte?
Implant anterior tissue from wild type oocyte into the bicoid mutant
Get the development of some anterior structures
How can ectopic transplantation of bicoid lead to bilateral symmetry?
If you take anterior tissue from wild type oocyte and implant in the middle of a bicoid mutant then you get bilaterally symmetry as thoracic segments all face the middle ectopic head
How can changing the number of gene copied of bicoid effect the developing oocyte?
If there are 0 copies then results in an oocyte with only 5 segments that are spread throughout the whole embryo (posterior)
If 1 gene (normal bicoid levels) then there are 7 segments and the original five or more posterior
If 4 genes (too much bicoid) then it compresses the segments posteriorly
How does bicoid act bind to DNA and act as a morphogen
High affinity binding site: activated at lower threshold concentrations of bicoid
Low affinity biding cites need high concs of bicoid to be activated
What is the role of pair rule genes?
Controlled by gap genes on a stripe by stripe basis
Kruppel and giant are repressors and bicoid and hunchback are activators
Each stripe is activated dependant on the interaction of positively and negatively acting transcriptional regulators
What is the role of segment polarity genes?
At this stage there is 14 segments but they need to be put together and show polarity within the segment (hairy cuticle and naked cuticle)
What is the relationship between Hh and Wg?
Hh and Wg feedback onto each other to maintain each others expression and refine segment borders
How is asymmetric pattern formed between Hh and Wg in the drosophila?
Hh maintains Wg expression which suppresses denticle development but an asymmetric pattern is formed because hedgehog causes degradation of wingless in posterior cells but not in the anterior cells creating a steep gradient on the posterior side
What is the role of hox genes in the drosophila?
Provide ‘who am I’ information to each segment.
Expression of homeotic genes along the a/p body axis occurs in the same order as the genes are within the genome.
Controlled by a combination of gap and pair-rule genes
Homeobox containing, DNA binding, transcription factors
How are hot genes laid out in the drosophila?
Two clusters
Patterned from anterior to posterior
What occurs when the antennapedia gene is mutated?
Mutation in drosophila hox genes that causes the antenna to turn to legs
Antennapedia gene is expressed in head as well as thoracic segments
What is meant by drosophila being a long germ band insect?
All 14 segments are defined at once
Meaning embryogenesis is complete in just 24 hours
When was the hedgehog gene discovered?
1980s
Name an animal that doesn’t have hedgehog signalling?
C.elegans
How many hedgehog genes are there in drosophila?
Hedgehog (Hh)
How many hedgehog genes are there in vertebrates?
Sonic Hedgehog (Shh) Indian Hedgehog (Ihh) Desert Hedgehog (Dhh)
Why is there more hedgehog genes in vertebrates then drosophila?
The genomes of vertebrates have undergone a number of genome duplications early in their evolution. As a result for several genes that are present in drosophila have more than one homolog.
How is the hedgehog ligand formed?
- Hedgehog genes are transcribed
- N-terminal region targets them the secretory pathway.
- The signal sequence is removed and then the protein undergoes an autoproteolytic cleavage catalysed by the C-terminal part of the protein (this c terminal part has no other role than promoting this cleavage).
- N-terminal part is coupled to a cholesterol molecule
- A palmitoyl group is added to the N terminus by the skinny hedgehog gene in drosophila and Hhat in vertebrates
- Both cholesterol and the palmitate are strongly hydrophobic and will render hedgehog quite insoluble in water and target it to membranes.
How is the hedgehog ligand diffused?
The hydrophobicity of the signaling component would make it impossible for the molecule to leave the cell membrane so it therefore requires the Dispatched protein
- a 12 transmembrane protien
- requires Scube glycoproteins
- Mechanism not understood
- likely to create multimeric hedgehog signaling particles that have their hydrophobic tail buried inside the particle or help load hedgehog molecules on lipoprotein particles
What receptors for Hedgehog are present in drosophila and vertebrates?
Drosophila - Patched - Smoothened Vertebrates - Patched 1 and 2 - Smoothened - Hedgehog interacting protein (Hhip)
What is the overview of how Hedgehog signalling works?
Patched continuously inhibits smoothened by stopping it from reaching the membrane by sending it for degradation
When Hh is present, it relieves the inhibition allowing smoothened to be active
What is the ration of inhibition of patched to smoothened?
Not 1:1
one patched can inhibit many smoothened
What is different about the mammalian hedgehog pathway?
It takes place in the cilia
- discovered as cilia mutants had the same phenotype as hedgehog mutants
How is it thought that patch works?
It acts as a pump
What does the patch receptor have homology too?
- Prokaryotic RND (resistance-nodulation division) permeases confer multi-drug resistance by pumping out toxins
- NPC1 (Niemann-Pick protein C1), which promotes the movement of cholesterol-laden vesicles along microtubules and can transfer some molecules across membranes
What is the protein NPC1?
Ptc is also related to NPC1, the gene mutated in Niemann-Pick type C1 disease.
Niemann-Pick disease (NP) refers to a group of inherited metabolic disorders known as the leukodystrophies or lipid storage diseases in which harmful quantities of a fatty substance (lipids) accumulate in the spleen, liver, lungs, bone marrow, and the brain
What is the role of the hedgehog interacting protein (Hhip)?
Hedgehog interacting protein is a vertebrate specific molecule that most likely acts to “mop up” free hedgehog thereby preventing it from reaching the patched receptor and thus keeping the signal down
How is patch hedgehog signalling promoted by other proteins?
CDO BOC and and the verebrate specific gene Gas1 most likely act as a co-receptor promoting the binding of hedgehog signal to patched and thereby promoting signaling.
What occurs in drosophila when no Hedgehog signalling is present?
In the absence of hedgehog, patched inhibits the activity of smoothened and two complexes exist that keep the transcription facor Ci reponsible for the effects of hh signaling out of the nucleus
- One containing costal2 (a kinesin like molecule that acts a scaffold protein) and fused a serine threonine kinase
- The other contains the Ci and supressor of fused gene (sufu) a gene without clear domains
- Under the influence of the first complex that is bound to Smo three other genes can act on Ci. They form complex consisting of casein kinase I Protein kinase A and Glycogen synthase kinase 3 beta.
- The transcription factor Ci which as a full length gene is a transcritional activator is processed under the influence of these genes to a shorter form via Slimb.
- The short form acts as a transcriptional repressor and is called CiR. In this way hedgehog target genes are actively repressed.
What occurs in drosophila when low Hh signalling is present?
When low concentrations of hedgehog are present it is thought that the PKA/GSK3/CKI complex dissociates from the complex containing Ci and as a final result, active repression by CiR is lost
What occurs in drosophila when high Hh signalling is present?
- At high concentrations, hedgehog acts through both complexes to release a fulllength Ci that will actively promote transcription of target genes
- This activation could involve phosphorylation but the exact nature if this phosphorylation is unknown
- it is thought that phosphorylation of sufu by fused promotes formation of the active form of Ci.
How is the repressor form of Ci in hedgehog signalling formed?
PKA phosphorylates Ci first, priming phosphorylation by GSK3 and CK1.
Mutation of any of these sites reduces processing to Ci 75, and reduces Slimb binding
Slimb is an F box protein, a component of the SCF ubiquitin E3 ligase complex
What are the feedback effect of Hh signalling?
Negative
- One of the best known targets of Hh signaling is Patched1/Patched - limit level of activation and reduces the range of movement of the Hh signal
- CDO/BOC/GAS-1 (positive regulators) downregulated
Postive
- Gli1 (not Ci), which is always an activator of transcription, is induced
Give examples of non canonical Hh signalling
In myocytes/adipocytes
- Ca2+ acts on smoothened and activating AMPK
- Causes cellular metabolism stimulating aerobic glycolysis, where glucose is used as an energy source but without fully “burning” it in the mitochondria
- leads to the production of lactate, which leaves the cell and will acidify the extracellular medium.
In drosophila embryo
- Wg and Hh maintain each others expression in an autoregulatory loop
In wing patterning
- Diffuses to anterior side and produced app at the boundary between cells
Neural development
- Different cell types are induced depending on the amount and duration of the hedgehog signal.
How can Hh signalling pathway lead to disease?
Hedgehog loss of function
- holoprosencephaly, caused by a loss of ventral brain structures
- cyclopamine
polydactyly (extra digits) and syndactyly (webbed digits)
How can Hh signalling pathway lead to cancer?
Gain of Hh signaling
-Basal cell carcinoma (BCC),
medulloblastoma rhabdomyosarcoma
-Inactivation of Ptc1 or Sufu: tumor suppressor genes
-Activating mutations of Smo: Smo is a proto oncogene
BCC most common form of cancer
What other forms of cancer can be caused by hedgehog signalling?
In other cancers, Hh acts as a growth factor, but the pathway is not mutated
- small cell lung carcinoma (SCLC)
- pancreatic cancer
- metastatic prostate cancer
What induces the organiser?
By signals released from the niewkoop centre
This activates BMP inhibitors
What is the pathway for ectoderm differentiating into epidermis?
- BMP binds to BMP receptor and a secondary signal (Smad) is phosphorylated
- Smad can then up regulate transcription factors like Msx1, GATA1 which leads to epidermal differentiation
What is the pathway for ectoderm differentiating into neural tissue?
- BMP inhibited
- No Smad phosphorylated so different transcription factors are up regulated (xlpou2, soxd)
- Neural differentiation pathway
What occurs where BMP is inhibited in the mesoderm?
Somite formation
What is neurulation?
When the neural plate rolls up to form the neural tube
What causes the formation of the dorso-ventral axis?
Establishes in response to diffusible molecules
- BMPs released from surface ectoderm (dorsally) and Shh released from mesoderm ventrally
What causes the early neural border cells to be established?
Intermediate levels of BMP signalling triggering transcription factors (msx)
How is it thought that BMP’s induce a different dorsal cell type?
- It was thought that they act as morphogens
- But recent work shows that roof plates may express many different BMP’s which induce a particular dorsal cell type
What are the key stages in ventralisation?
- Notochord secretes shh
- shh diffuses through spinal cord - conc gradient (morphogen)
- It induces different patterns of transcription factors
- High concs of shh cause floor plate cells to develop which occupy ventral midline of neural tube - activates shh
- shh mRNA in both notochord and floor plate
- shh diffuses into neighbouring cells causing ventral fate
What occurs during gastrulation?
- The epiblast cells converge towards the primitive streak and involute down the streak
- Epithelial cells transform to mesenchyme which is important in the ability of cells to migrate
- Endoderm cells go deeper and mesoderm stays between endoderm and epiblast cells
- Formation of mesoderm
Where is the paraxial mesoderm located and what does it form?
It is posterior to Hensen’s node and forms the somites
Where is the lateral mesoderm located and what does it form?
Found laterally to the axial mesoderm
Responsible for limb and heart formation
What does the intermediate mesoderm form?
Contributes towards formation of urogenital system - kidney and gonads
What does axial mesoderm form?
The notochord and precordal mesoderm
What are are somites?
Perfectly symmetrical small cells on either side of the spinal cord
- Show evidence of segmentation in invertebrates
- Segmented paraxial mesoderm
Give an example of segmentation in vertebrates
Somite number dictates number of spinal vertebrae
- Humans have 33 vertebrae at adulthood
- Human embryo has 38-44 somites
When is the primitive streak no longer present?
When somites no longer form - number of somites is fixed for a given species
