What are the two opposing theories of plant development?
- Preformationism= Nicolas Hartsoeker (1695)- organisms develop from miniature preformed versions of themselves, hommoculus
- Epigenesis= Aristotle- new structures slowly added to form whole body
The cell Theory- Robert crooke (1665)
- All organisms are composed of 1 or more cells
- Cell most basic unit structure
- All cells arise from pre-existing cells
What is germ plasm determinants theory?
Fredrick Weisman 1880s
Germ cells have whole collection of somatic cells which have subset of determinants.
Carry out specific characteristics determined
Wilhem Roux- 1888
Killed one half of frog egg with hot needle.
Half remains dead the other half embryo
Separated embryo at stage 2 and 4
both reform themselves and fully develop
NOT in contact with each other, disrupt communication and modify same outcome
How do cells aquire new properties that differentiate them from others?
Cell division- symmetric or asymmetric
Cell adhesion- condensation or dispersal
Cell shape-epithelial or epithelial to mesenchymal
What are the different types of cell-cell communication
Paracrine= Signal produced in one cell and binds to another
Autocrine= provides on one cell, acts on other side of same cell
WNT, SHh, TGF, BMP signalling ^both above
Juxtacrine= cell/cell contact
Cell signalling- cell-cell interaction
Cell needs to be competent- receptor for signal and machinery
signal can be instructive (initiates new programme, direct change in cell) or permissive (changing environment for another to react)
signal can act as morphogen
What does transmission of signal to the nucleus involve
Signal transduction cascade
What are the 3 germ layers?
- Ectoderm (external layer)= Skin, brain/neurons and pigment cells
- Mesoderm (middle)- Cardiac, skeletal, RBC, smooth muscle
- Endoderm( Internal layer)= Lung alveoli, thyroid and pancreatic cell
What 2 methods is tissue homeostasis maintained by?
- Negative feedback loop
2. Stem cell mediated repair
What is pattern formation in embryonic development?
Process where cells are ordered within embryo.
Need Co-ordinates to establish axis
What is morphogenesis?
Cell and tissue movement and behaviour changes in cell
Factors affecting morphogenesis of cell?
Step by step series of differentiation of cell in embryonic development
- Specification- early commitment, not stable
- determination- commitment to cell fate, stable , still aquisation of terminal characteristics
- Differentiation- reversible
- Maturation- irreversible
What are the processing underlying embryonic development?
Growth of embryonic development
- continuous process
- growth rate varies depending on age
- cell proliferation, enlargement and accretion
What are the two opposing theories in early embryonic development in vertebrates?
- Funnel (Haeckal)= Few differentiate in early development X wrong
- Hourglass (van baer)= Lots of differentiation beginning and end Correct
Methods to find out where and when a gene is expressed in the embryo
- In situ Hybridisation
- Northern blott
- reporter lines (transgenic)
What is a method to find out how many transcript is in an embryo?
RT-PCR- carry out amplifcation of gene expression
Is the protein expressed with the same timing of the gene?
Look at mRNA and protein as the protein may not be expressed where it is transcribed
look at distribution of protein- western blot (how much protein made) or immunochemistry (temporal and spatial
To find out if the gene/ protein is essential for development
Loss of function- disrupts expression
gain of function- increase amount of activity to gene product encoded by mutant gene
Forward and reverse genetics
C.elegans, drosophilla, mouse and zebrafish
Seeks to identify gene whose mutant causes a specific phenotype.
Cross mutant and WT- F3 generation gives m/m (mutant), m/+, +/+
Target 1 gene you are interested knowing function of by two main methods:
- Straight KO
- Conditional KO
- homologous recombination(loxp)
- Es cells transfection
- ES cell selection
- Es cell injection into blastocyst
- Implant into mouse
- Selection of chimeric mouse
- breed either conventional KO or conditional KO (floxed allele with tissue specific cre mouse)
How is a gene regulated?
Tissue manipulation- drugs transfection and electroporation
- Tissue ablation, graft and transplantation. (surgical)
- Bead/ cell implantation (signalling molecules, drugs)
Observational biology to see use of gene, experiment in chick and quail
Inject quail into chick Carry out early in devel when cells aren't committed to fate Use antibody to recognise quail cells Precisely trace all 300 Basic genetic construct now possible
What is a morphogen?
Soluble secreted molecule that acts from a distance to specify the fate of cells.
Not all molecules that are involved in patterning are morphogens
Need to make and break otherwise lose info
What must a morphogen do?
- Induce different outcomes at different concs
Instructive-provide info to cell next to it
permissive- Have knowledge about what they are/gonna be PERMISSIVE NOT MORPHOGEN
- Act directly at a distance and NOT be diffusible through cells (bucket brigade)
Tests to show that permissive are not morphogens for
- Provide a second source of signal in ectopic expression- opposite side
- instructive would get mirror image
- permissive no effect
- provide red signal at a uniform conc
- instructive all see same gradient of signal so all same
- permissive no effect
Test for morphogen acting directly at a distance
- Use genetic engineering to make the proposed morphogen a juxtacrine (eg add a trans membrane domain)
Stop peptide from diffusing
Morphogen - adjacent cell to signal will see it but none of the others
- Make genetic mosaic that lacks signal
Binding of molecules in extracellular matrix and high conc of receptor can generate a steep gradient,
What are HSPGs?
Heparan sulfate proteoglycons found in extracellular matrix and bind to ligands Also known as co-receptors Regulate morphogen diffusion by: - slowing diffusion (BMP) -facilitating diffusion (Hedgehog)
How does planar transcytosis play an important role in establishing morphogen gradient?
Repeated endocytosis and restriction of morphogens allow it to travel through the cells
Evidence= antibody staining shows dpp found in vesicles and mutations that block vesicle formation cause dpp to act in a juxtacrine manner
How do cells read or interpret a gradient to make a cell fate decision?
Transcriptional read out model
- Higher conc= higher conc of activated TF
- More TF enter nucleus and direct transcription
Bicoid= morphogen and TF
-accumulates in nuclei of synaptical blastoderm generating conc gradient
How is TF concentration interpreted at the DNA level?
Enhancer have different affinities *medium TF concentration low affinity=OFF High affinity= ON Even though both recieve same conc only high affinity enhancers can bind enough to TF to cause gene expression *Low TF conc Low affinity=ON High affinty =ON
How are strict thresholds achieved when concentration is not steep?
The lifecycle of drosophilla melanogaster
- fertilised egg
- larva- pupa
History of drosophila
1910- morgan discovers a while eye fly (genetic map formed
1913- Sturtevant constructs first genetic map
1914/16- show chromosomes must contain genes
1927- muller shows x-rays cause mutations
1979/80- saturation mutagenesis to identify genes involved in development
1980-00s- p-element, enhancer trap, gal 4/UA5 missexpression, FLP/FRT clonal mutant anaylsis
2000- Genome mapped!!
Drosophilla courtships- what are the genetically encoded behaviours?
- Wing vibration
- Attempting copulation
what is the timeline of development of drosophilla egg?
- Stem cell maintenance= divide and move away fro source, 4 incomplete cytoblast mitotic divisions
- 2 nurse cells and 2 have full connections around (1 becomes egg)
- Selection of future oocyte:
- nurse cell= endo-replication (DNA replication without cytokinesis, many copies of genome
- Oocyte= diploid nucleus or meiotic recombination
what is the subcellular localisation of maternal factors?
A/P Localised by microtubule transport minus and plus ended motors glue anchors them in position waterproof and sealed
What is chorion?
Egg shell secreted by follicle cell
What is the vitelline membrane?
Hydrophobic, protects from drying out
How do you transform a single cell into an embryo?
- Set aside ‘top’ cells versues ‘bottom’ cells by differentiation
- ‘Bottom’ cells signal to ‘top’ cells so that the latter start to differentiate further forming 3 germ layers
What is cell differentiation?
Process by which cells become different from each other and acquire specialised proteins. Governed by changes in gene expression which dictate protein synthesis
How is gene expression governed in a cell?
Intrinsic and extrinsic factors
What are the top and bottom layer in xenopus and chick/humans?
Bottom= Hypoblast `
How does top/bottom layers lead to body axis?
Initiate early differences in future top (vegital) and bottom (animal) hemispheres.
Due to gravity in xenopus, different cytoplasmic determinants have sunk to one part of egg so oocyte is polarised before fertilisation.
As mitosis continues an early cleavage further separates vegital and animal so cytoplasmic factors with were on vegital side are now resticted to this hemisphere.
Cytoplasmic factors all act on DNA to cause gene expression
What causes formation of blastocoel?
Changes in osmolarity when the cell continues to divide so hollow ball of cells is composed
How many days does it take for the embryo to be implanted into the uterine wall?
12- the morcula separaated into top epiblast and bottom hypoblast
What is the evidence animal is different to vegetal?
TF VgT localised to the nucleus of the vegetal hemisphere binds to promoter and activates transcription of nodal (codes for a secreted morphogen- protein that can diffuse out of vegetal cells into animal)
In situ hybridisation
How is a 3rd germ layer produced?
Animal hemisphere cells which contain receptors for nodal-the signal transduction pathway is actiavted
Causes change in behaviour- cell proliferation, migration, differentiation
What happens to cells with different levels of nodal signalling?
Where is VT and nodal expressed in a chick/human?
Expressed in posterior part, cells responding to nodal migrate to the centre, drop and reform
Wnt signalling pathway is activated on dorsal side of embryo and gastrulation starts
Where is the sperm entry point?
Where the future organiser will form.
Opposite side to dorsal
Initiates rotation and 2nd signal pathway for wnt
What is the nieuwkoop loop?
region in vegetal hemisphere where the nodal and beta catenin overlap
Beta catenin causes nodal activity to be transformed into gradient and elicts different cell fates
Why does the combination of high nodal and active wnt signalling induce the organiser?
Some genes like Gsc are only transcriptionally activated via BOTH nodal downstream effectors and wnt/beta downstream effectors
What happens to cells in the nucleus of the organiser/node which express TF?
Bind to promoters to alter behaviour
Organiser/node differentiates into axial mesoderm
Cells migrate through convergent extension
Why cells in the node/organiser differentiate into different types of mesoderm?
Slightly different levels of goosecoid and saimois expressed so form prechordal and notochord
How does anterior/ posterior axis formation occur?
Organiser undergoes self-differentation
organiser composed of a mix of progenitor cells
prechordal mesoderm to form axial mesoderm
organiser undergoes convergent extension
Three main cell type ‘precursors’ form in the organiser region?
Organiser cells coalesce and form…..?
Rod, which migrates inside the cell of animal hemisphere on top of blastoceol, follow fibrnection-rich pathway, binds integrins expressed due to organiser.
Migration order of the rod?
Pharyngeal, prechordal (short) then notochaord (long)
What does the axial mesoderm underlie?
Prospective neural tissue
What signals form A/P identity?
Posterior identity= FGF and RA
Anterior Idnetity= Anti-wnts
What meditates neuralisation of ectoderm and dorsalisation of mesoderm?
Bmp antagonists- secreted molecules expressed by organiser eg. Chordin
Vetebrate proto-oncogene that encodes a mouse homologue of wingless
How does wingless maintain its expression?
Wg and Hh maintain each others expression in an autoregulatory loop, Hh upregulates wg but wg controls engrailed (En) a TF which regulates Hh expression.
What are the factors in generating wnt?
- Several wingless like genes
- Vertebrates have more wnt genes than drosophilla due to the genome duplication during evolution.
How is wnt formed?
Produced in a signalling sequence which is cleaved off when it enters the secretory pathway
Acetyl-transferase required for the formation of wnt signal
Porcupine gene= adds one or both modifications to the wnt protein
What is the wnt receptor?
Protein made up of frizzled and LRP/arrow
Binds to N terminal , cysteine rich domain
LRP/arrow is also a single pass transmembrane which makes contact with wnt
What is an extra cellular wnt inhibitor?
DKK1 (dickopf1) binds to LRP and kremlin promoting the internalisation of LRP
Used to down regulate wnt
What is the canonical signal reception transduction?
When wnt is absent
Absence of wnt
-b catenin is phosphorylated by destruction complex (axin, binds to APC, b catenin and 2 kinases (GSK3beta and CKLa)
- phosphorylated b catenin is recognised by slimban f box/WD40 repeat protein causing b catenin to be ubiquinated and degraded
-enzyme is proteosome
-absence of b catenin, bound to promoter of wnt responsive genes associated with Groucho
What’s the canonical signal reception transduction? When wnt is present?
- wnt binds to dishevelled is recruited to frizzled which is then phosphorylated
- Arrow is phosphorylated by gsk3 and Axin is recruites to the receptor
- Slimb is lost from complex
- inactivation of destruction complex
- b caterin will accumulate and not broken down
How is beta catenin degraded?
Phosphorylated by ckla Which primes phosphorylation by gsk3
Posphorylated by both kinases is required for beta catenin recognition by an E3 ubiquitin ligase complex and subsequent recognition by the proteosome
Serine/thryosine phosphate form optium binding site for B-trcp
How is beta-catenin phosphorylated?
CK1 binds to the N terminal on the b-catenin priming phosphorylation by GSK3 at site
Slimb/b-trcp binds after Gsk3 phosphorylates the 3rd/4th phosphorylation site
B-Trcp/slimb and SCF E3 ligase, what is their role in the signalling pathway?
SCF E3 ubiquitin ligase complex contains ROC which binds to E2
Fbox protein interacts with SKP1 via its F box and with the substrate via another domain that interacts specifically with phosphoryated targets
B-trcp is made up of WD40 protein (repeats of beta-trcp interact with specific substrates B-catenin and ci)
What does groucho do?
Recruits histone deacytlases recruits Pygo (pygopus), lgs (legless) and BRG1 (chromatin remodelling) Mutations in the first 2 ^result in wingless like phenotype
What other pathways is wnt involved in?
Drosophila- ligand debated
Zebrafish- wnt11 and wnt5 mutations show effects on convergent extension
What are the roles of canonical wnt in different animals?
Drosophila- segementation - expressed at the d/v boundary of the wing required for patterning and outgrowth
C.elegans- regulation of neuronal fate and their migration
Zebrafish- early= nuclear b-catenin induces dorsal,wnt11
Late=wnt8/3 induces ventral/posterior fates (loss of anterior features, posterioristaion of brain)
Wnt and intestinal stem cells
Small intestine contains crypts (paneth cell) and villi (enterocytes, enterendocrine and globlet cells)
Epithelium of both intestines is continuously renewed by stem division
SC slowing divide but rapidly produce dividing transit amplifying cells therefore creating a CONTINUOUS FLOW
wnt expressed by stroma below intestinal crypts-
Loss of TGF4 or overexpression of dk6 promotes sc loss so wnt required for maintenance
Wnt in cancer
Ectopic wnt signalling can occur by loss of APC (tumour suppressor )
- familial adenomatus polyposis
-many polyps in colon and rectum
-occasional loss of remaining APC (active wnt increased proliferation)= malignant tumour
Gain-of -function wnt= Beta-catenin and APC-sporadic colon cancer
Loss-if-function= Hepaocellular carcinoma
How is the fruit fly segmented?
Can be seen with denticle belts (hairy) and naked cuticle
head splits into T1,2,3
Tail splits A 1-8
Segmentation seen early after gastrulation
What is a complentation group?
Pairs of genes all cause embryonic genotypes but see if theyre in the same genes
Test to see if pair genes are in the same gene
Put 2 chromosomes together, If they show phenotype then they fail to complement
Set up matrix to compare
Oversampled to unlikely to see genotype they havent seen before
ONLY find what youre looking for
What are the classes of mutations for segmentation?
Knirps- removes chunk of organism
Paired- every other segment missing (1357 or 2468)
Gooseberry- miss half of each segment, delete naked cuticle
What is bicoid and what does it do?
Bicoid= maternal gene, DNA binding TF
Maternal gene-gap gene - pair gene- segment polarity
=Morphogen- only one direct TF
located in anterior embryo
Moves into nuclei as DNA binding
maternally loaded into developing ooctye
Testing to see if mutant has loss of anterior structure?
Take cytoplasm from wild type and put into bicoid of mutant egg then you can partially rescue
Morphogen has ability to pattern independently
Transplant into middle= ectopic head structures and mirror image thoracic segments
Results of segmentation pattern
- 5 markers present (mutant)
- 7 markers present (WT)
- All posterior segments- bicoid conc moved down embryo
High affinity for binding site then activated at lower bicoid conc
Low affinity for binding site then activated at High bicoic conc
Wingless and hedgehog expression
Wingless= hairy cuticle
Hedgehog= naked cuticle segment (shh present)
feeback loop for each other
Hh upregulates wingless with activates Engrailed which upregulates Hh
Genes for segmentation
Gap- control pair genes
Pair rule genes- controlled stripe by stripe, dependent on the interaction of positive and negative acting transcriptional regulators
Segment polarity genes- divided into 14 segments, genes pattern into hairy and naked
What is a hox gene?
Provide who am i info in each segment
Expression of homeotic genes along A/P axis
Controlled by combo of gap/ pair rule genes
What are short, intermediate and long germ band insects?
Long=Drosophila (14 segments all defined at once)
- quick (24hrs)
- complicated (maternal, gap, pair and segment in each segment)
Short/Intermediate= start with head then add abdominal (posterior discs appear to bud off)
The segmentation clock
Notch( receptor) delta (ligand)
notch activation causes down regulation of notch ligand
time lag in responses causes oscilations between strong and weak signalling
propagation between cells causes wave of activation
Hh signalling ligand that have been found?
Responsible for Hh ligand formation and for its diffusion into neighbouring cells
How is Hh sginal formed?
- Hh genes transcribed with N terminal signal sequence that targets secretory pathway
- signals sequence removed and C terminal part of protein cause autoproteolytic cleavage
- At same time N terminal is is coupled to cholesterol palmitoyl by Ski (skinny Hh)
4.Both Cholesterol and palmitate= hydrophobic and make Hh insoluble to water/ target molecule
Hyrophobic- impossible to diffuse form cell membrane so it can only signal to neighbouring cells
- Dispatched (12 tm proteins) and secreted scube protein are required for release of signalling cell and help create multimeric Hh signalling protein
- Hspg’s required for long range diffusion of Hh particles
What happens when the signal has been produced?
It has to be perceived by cells that are competent to respond to signal
2 crucial transmembrane proteins in Hh signal pathway?
Patched= 12tm protein which binds to Hh acts in negative way as it binds smoothened when ligand is absent Smoothened= 7TM protein
Ptc inhibits Smo
When Hh binds then Smo is relived
What did experiments on drosphila show about ptc and smo?
Show Ptc and Smo do NOT form schoichiometric complex
Single ptc can inhibit large number of smo
ptc regulates subcellular and stability of smo
What happens in the absence of Hh?
Ptc keeps smo away from the cell surface
regulat trafficking of smo to place where it gets degraded.
When Hh there they both get internalised and degraded
What are the 3 changes that occur to smo?
Why is mammals cillia a focal point for Hh sensing?
No Hh signal- signal ptc1 is localised to the cilium of the cell and smo is excluded from this territory
Hh signal-binds to ptc removing ptc1 from cilium allowing smo to accumulate and initiate signalling
Is ptc a pump?
Ptc has homology to prokaryotic RND (Resistance- nodulation division) permeases confer multi-drug resistance by pumping out toxins
NPCl (group of inherited disorders) promotes movement of cholesterol-laden vesicles along micro tubules and can transfer some molecules across membrane
Ptc like proteins in C.elegans may be involved in membrane remodelling efflux of lipid/lipid modifies molecules.
-ptc may pump small molecules into cells that inhibit smo or pump out cells that activate smo
What is Hedgehog interacting protein (Hhip)?
In vertebrates specific molecules that mops up free hedgehog thereby preventing it from reaching ptc receptor so keeps signal down
What are the 2 complexes that exist to keep TF ci responsible for the effects of Hh signalling out of the nucleus in the absence of Hh?
- Costal 2- a kinesin like molecule that acts as a scaffold protein and fused a serine threonine kinase
- Ci and suppressor of fused gene (sufu) contained a gene without clear domains
- form complex with pKa,Gsk3 and Ck1
-TF ci (a trascriptional activator) processed under genes to form a shorter form via slimb
short form= Ci receptor (CiR)
What happens with different concs of Hh?
Low Hh- PkA/Gsk3/Ck1 complex dissociates from complex containing ci and as a result active repression of CiR is lost
High Hh- acts through both complexes to release a full length Ci that will actively promote transcription of target genes
Ci Phosphorylation and Proteolysis
In absence of Hh Ci is processed to shorter form
Pka, Gsk3 and Ck1 phosphorylation are required for cleavage
Slimb binds to a combination of non-optimal binding sites
the proteosome partially proteolyzes ci leaving the N terminus intact
The proteosome usually completely degrades substrates
How are PkA Gsk3 and CK1 conserved in Ci and Gli?
Gli1 doesn’t get cleaved to a shorter repressor form and acts only as a transcriptional activator
Gli2 and 3= activators and repressors and contain an extra PkA site
Ci- the PkA Gsk3 and CK1 sites conserved in vertebrate homologues
High Hh=PkA functions, when no PkA then Gli and Ci are no longer proteolyzed
What are the negative feedback effects of Hh signalling?
Ptc/Ptc1 limit level of activation and reduce range of movement of the Hh signal
What are the positive feedback effects on Hh signalling?
Gli an activator of transcription induced
What are the non canonical Hh signalling?
- Smo-Ci-RhoA endothelial cells in cytoskeleton
- Shh boost Ca2+ and IP3 signals in spinal neurons
- myocytes/ adiopocytes-shh activates smo- ca2+-AmpK signalling
How is Hh expressed in the wing?
Expressed posterior and diffuses anterior where it induces decephentaplegic (signalling molecule related to BMP/TGF b helps to pattern wing)
How is Hh expressed in neural development?
Neural tube cells develop into different types of neurons depending on how much Shh they receive
vertebrates notochord and floorplate together make the neural tube
How does Hh effect AP patterning of the limb?
Hh is active in posterior limb bud where it forms the zone of polarising activity a region that can give posterior identity to the forming limb and also involved in outgrowth.
How does Hh cause disease?
Development -Loss of Hh- haloprosencephaly (loss of ventral brain) -cyclopamine = inhibits smo -polydactly= extra digits -syndactyly= webbed digits Cancer -direct result of Hh activation 1. Basal cell carcinoma 2. medulloblastoma 3. Rhobdomyosarcoma -inactivation of PK1 or Sufu which are tumour suppressor genes -smo= proto oncogene so activating mutant -loss of ptc- BCC
What is the smo cancer treatment?
- strong Hh inhibitor
- few months it comes back and is worse