Basic principles and techniques Flashcards
Where does nearly every cell of the body come from?
Which cells are the exceptions?
Come from the 3 germ layers
With the exception of germ cells
When are germ cells laid down?
During gastrulation
What happens to germ cells?
They are set aside during development and follow their OWN developmental pathway
What does the ectoderm give rise to?
The outside layers and the development of the nervous system:
- Skin
- Epidermis
- Neurons in brain
- Pigment cells
What does the endoderm give rise to?
Inner organs:
- Alveolar cells
- Thyroid cells
- Pancreatic cells
What does the mesoderm give rise to?
- Muscle
- RBS
- Tubule of kidney
What is tissue homeostasis essential for?
Normal development, growth and repair of organs
What is tissue homeostasis?
The balance of production of new cells (tissue growth) and the cells lost through death and damage
How is tissue homeostasis controlled?
Describe this
Through a negative feedback loop:
- Process occurs until a certain point where a specific factor is produced
- This factor blocks the process through blocking cell signalling or blocking gene transcription
What process is heavily used in developmental biology as a regulatory mechanism?
Negative feedback
What is another way to control tissue homeostasis?
Describe this process
Stem cell-mediated repair:
- Produce new cells to replenish the tissues that have been lost due to death and damage
Is stem cell-mediated repair fast or slow?
Rapid is some tissues (eg. skin)
Slow in other tissues (eg. bones)
What does disruption in tissue homeostasis cause?
How?
Disease:
- Excess progenitors –> cancer
Ageing:
- Excess differentiation/death –> tissue degeneration/death
What 4 processes underlie embryonic development?
1) Pattern formation
2) Morphogenesis
3) Cell differentiation
4) Growth
What is the definition of pattern formation?
- The process by which cells are organised in SPACE and TIME
- To produce a WELL-ORDERED structure within an embryo
How are cells organised in space and time?
Cells at any time/position in the embryo are defined by 3 coordinates
What does the positional information of a cell tell the cell?
WHAT TISSUE it belongs to and WHAT BEHAVIOUR/cell fate it should have
Is positional information an intrinsic of extrinsic property of the cell?
Intrinsic property
What is needed for the cell to have positional information?
Establishment of 3 axis:
- AP
- LR
- DV
What order are the 3 axes of the body laid down?
- Anterior/posterior axis
- Left/right axis
- Dorsal/vental axis
What is the definition of morphogenesis?
Cell and tissue MOVEMENT and changes in BEHAVIOUR that give the developing organ its 3D shape
What are 4 processes that occur in morphogenesis?
1) Cell adhesion
2) Cell migration
3) Cell death
4) Cell shape
What does changes in cell shape facilitate?
Migration
What is cell differentiation?
Process by which cells become DIFFERENT to each other and ACQUIRE specialised PROPERTIES
What is cell differentiation governed by?
Changes in GENE EXPRESSION
That dictate the REPERTOIRE of PROTEIN synthesised
What does the repertoire of proteins in a cell dictate?
The FUNCTION of the cell
How does differentiation proceed?
Over time:
- Cells GRADUALLY acquire more and more specialised properties
- And DECREASE in pluripotency
What are the stages of differentiation?
1) Egg/stem cell - pluripotent
2) Specification
3) Determination
4) Differentiation
What developmental stage comes after differentiation?
Maturation
In a tissue, in a specific cell lineage, what can be identified?
Cell intermediates for each stage of development:
- Specification
- Determination
- Differentiation
- Maturation
What is specification?
The UNSTABLE engagement into a certain cell lineage
What is specification dependant on?
The CONTINUOUS stimulation by the environment or intrinsic cues
How can specification/determination be tested?
Transplant the cells into a different environment:
- If the cells change characteristic - commitment isn’t stable
- If the cells continue to differentiate in the normal way - the cells are DETERMINED
What is growth?
Increase in mass or size
Continuous process
What does the growth rate depend on?
Age and organ
What does growth include?
- Cell proliferation
- Cell enlargement
- Accretion
What is accretion?
The addition of extracellular matrix around the cell to give the cell a larger size
How can we study the changes in gene expression profile, and cell behaviour and cell-cell communication?
1) Embryology
2) Developmental biology
3) Animal models/use of genetics
What is embyrology?
OBSERVATIONAL biology and experimental MANIPULATION
What is the study of developmental biology?
Study of GENE and PROTEINS
How can animal models be used to gain knowledge of how the HUMAN develops?
EARLY embryonic development of VERTEBRATES share many similar features
What are the 2 lead models of using animal models?
1) Funnel model
2) Hourglass model
Who proposed the funnel model?
Haeckel
What is the funnel model?
- Embryonic development between organisms is highly similar in the VERY EARLY stages of development
- Diversity occurs when reach LATER stages of development
Who proposed the hourglass model?
Von Baer
What is the hourglass model?
VERY early embryonic development - not similar (eg. gastrulation)
BUT, the intermediate stages of embryonic development are VERY similar
How can animal models be used to study diseases of humans?
- Many genes responsible for human diseases cause similar effects in model organisms
- Can cause the disease in the animal model and apply the findings to the human
What 5 questions must be answered in order to determine if a newly discovered transcription factor is important in embryonic development?
1) WHEN and WHERE is the gene transcribed in the embryo (expressed)
2) Is the protein expressed with the SAME TIMING as the gene?
3) Is the gene/protein ESSENTIAL for development?
4) How is this gene regulated?
5) What are the tissues/organs that derive from the cells that express this gene
To see if a gene is being transcribed, what must be detected?
Detect the presence of the mRNA of the gene
What 5 methods can detect if the mRNA of a gene is present?
1) In situ hybridisation
2) NORTHERN blot
3) RT-PCR
4) Micro-array
5) Transgenic reporter lines
Which methods of detecting mRNA allow you to detect WHERE the mRNA is present?
- In situ hybridisation
- Northern blot
- rt-PCR
- Reporter lines
What is the condition of using northern blotting to detect where the mRNAs are present?
Can only detect where they are present if you can first isolate the organ before performing western blot
Describe the process of in situ hybridisation
- Generate a probe complimentary to the mRNA of interest - incorporate a compound into it
- Probe forms a hybrid with mRNA is present
- Detect where the probe is using an antibody that is specific to the compound incorporated into the probe
Couple the antibody to a detection system:
- Substrate for alkaline phosphatase on the antibody
- Add alkaline phosphatase - will remove a phosphate from the substrate and produce a blue colour where the antibody present
How can in situ hybridisation be used to see how gene expression changes over time?
Perform in situ hybridisation in embyros at different time points
What is the function of reporter lines?
To reveal where the gene is expressed in the embryo
Describe the process of reporter lines?
- Remove the gene of interest from the DNA sequence
- Replace the gene of interest with the sequence of a reporter gene (encodes GFP)
- Introduce this gene into an animal model
- This reporter gene is now under the influence of the genes that control the expression of the gene of interest
- Where the gene of interest is normally expressed - there is now expression of GFP
How is reporter line generation different to gene fusion with GFP?
Reporter line shows where the GENE is expressed (mRNA)
Gene fusion shows where the PROTEIN is in the cell
What is the function of microarrays?
To compare the expression of genes present in the genome of a cell compared to another
Eg. mutant, WT
Eg. treated, untreated
Describe the process of microarrays
- Isolate the transcript
- Reverse transcribe to form cDNA
- Label the cDNA with different fluorescent tags
(sample A= red, sample B=green) - On the grid - one spot represents ONE transcript (mRNA)
- Apply cDNA from each cell
If spot is red = only present in sample A
If spot is green = only present in sample B
If spot is yellow = present in BOTH sample A and sample B
Why is it important to see if the protein is expressed at the SAME position as the mRNA?
Site of expression of the protein may be very different to that of the transcript
Can give ideas on the function of the protein - eg. trafficking, morphogen, extracellular matrix protein)
What are 2 methods of protein detection?
1) Western blotting
2) ImmunoHISTOchemistry
3) Fusion protein
What is another name for immunohistochemistry?
Immunofluorescence
What is another name for western blotting?
Immuno blotting
Which methods of protein detection allow to see the DISTRIBUTION of the proteins in the cell?
Immmunohistochemistry
Fusion protein
Which method of protein detection required prior knowledge of the protein?
Why?
Immunohistochemistry
Need to make a specific primary antibody
What is the method of immunohistochemistry?
- Incubate tissue in the presence of a primary antibody
- To find the position of the primary antibody - add a secondary antibody with a fluorescent tag
Using immunohistochemistry, how can more than one protein be visualised at the same time?
Adding multiple specific primary antibodies
Adding multiple secondary antibodies with specific colours
Describe the process of fusion proteins
- Insert the DNA of GFP IN FRAME onto the end of the DNA sequence that encodes for the gene of interest
- Produces a SINGLE transcript
- Produces a single protein with the protein of interest fused to GFP
How can fusion proteins be used to visualise the position of the protein of interest?
The gene of interest with GFP is still controlled by regulatory sequences
Proteins are present in the places they usually are but can now be visualised as they express GFP
Can gene fusion be done in live animals?
Why?
Yes
GFP doesn’t alter the function of the protein
What tests can be done to determine the function of the gene?
- Gain-of-function experiments
- Loss-of-function experiments
What genetic techniques are used in loss-of-function mutations?
1) Forwards genetics
2) Reverse genetics
What is the difference between forwards and reverse genetics?
Forwards genetics:
- Trying to FIND gene of interest
- Randomly mutate the genome
- Find interesting phenotypes
- Find the genes responsible for these phenotypes
Reverse genetics
- Already KNOW gene of interest
- Eliminate gene of interest to look at the function
- Infer effect
What method is used for gain-of-function experiments?
Transgenesis
What are loss-of-function studies testing?
If the gene is necessary for a certain function
What are gain-of-function studies testing?
If the gene is sufficient for a certain function
What animal models are used for forwards genetics?
Why?
C.elegans
Drosophila
Zebrafish
Mouse
All have rapid generation times
What agent is used in forwards genetics?
Why?
Chemical mutagen (eg. ENU)
To randomly mutate the genome
In forwards genetics, what are most of the mutations formed upon exposure to a chemical mutagen?
How do we gain access to these mutations?
Most mutations are recessive
Gain access b:
1) Crossing a mutant animal with a WT animal - to form some WT and some carries
2) Incross these animals - generates 25% mutant, 25% WT, 50% carriers if a mutation is present
If there is a mutant phenotype during forwards genetics, what can be assumed?
That there has been a disruption of a gene that is important in the NORMAL development of the tissue of interest
After reverse genetics, what can be used to identify the gene mutated?
Positional analysis
What methods are used in reverse genetics?
Gene knock out
Gene knock down
What is gene knock out?
Elimination of the function of the gene in EVERY CELL of the body
How is gene knock out achieved?
- Insert sequence (eg. for antibiotic resistance) INSDIE the gene of interest - disrupts the expression of the gene
- Introduce construct back into ES cells
- Select which ES cells have taken up the construct using antibiotic selection
- Insert ES cells into the inner cell mass of the blastocyst of mouse embyro
- Reintroduce embyro into surrogate mother
- Breed the mice to form a mouse in which EVERY cell in its body has the mutation
What is the presence of successful integration of the construct into cells (in gene knock out method) indicated by?
Indicated by the level of chimerism of the mouse
What is the problem with gene knockout?
How can this be overcome?
Unable to look at genes that also has an essential function in early embryogenesis - embryos die very early on
Overcome by using CONDITIONAL KNOCK OUTS
What do conditional knockouts allow?
Ability to determine WHEN and WHERE the knockouts occur
What is the process of conditional knockouts?
1) Flank gene of interest with loxP sites
2) Insert into ES cells, into blastocyst, into surrogate
3) Produces a normal mouse that contains loxP sites on either side of the gene of interest - this mouse is normal
4) If cross the mouse with loxP sites with a mouse that expresses either:
- Cre recombinase under a TISSUE SPECIFIC promoter
OR - Cre recombinase under a promoter that can be controlled in a TEMPORAL manner by induction of a drug
If the cre recombinase is present under a tissue specific promoter, what happens when cross with a mouse with loxP sites?
Cre recombinase will only be expressed in that tissue
Gene only knocked out in that tissue
What are loxP sites?
Specific DNA sequences that are recognised by Cre recombinase
They are joined together in the presence of Cre recombinase - causing deletion of the gene
What are the methods that allow detection of how the gene of interest is regulated?
1) Embryology
2) Manipulation of signalling pathways
What is tissue manipulation in embryology used for?
To demonstrate the inductive function of a tissue
What are the methods of tissue manipulation?
- Tissue ablation
- Tissue grafting/transplantation
What methods are used to show the inductive function of a specific molecule?
Transplant a soaked bead/specific cell
What did Spemann and Mangold do?
- Transplanted a small region from the dorsal side of an embryo into a host embryo (on the ventral side)
- Observed axis duplication - transplanted region had organiser function
Are all animals suitable for embryological approaches to determine what regulates the gene?
Why?
No
Must consider the accessibility and size of the embryo (eg. mouse develops in the placenta)
What did John Saunders and Robert Riddle do?
- Transplanted the ZPA from the posterior region of the chick embryo, into the anterior region
- Saw duplication of the foot - this region had organiser function
How can we see what tissues and organs derive from the cells that express the gene of interest?
Fate mapping using:
1) Embryology
2) Genetics
What is observational biology used for?
- Fate maps
- Lineage analysis
What are 3 methods of observational biology?
1) Cell/tissue transplantation
2) Cell/tissue labelling with DYE
3) Cell/tissue labelling GENETICALLY (GFP, brainbow transgenic lines)
What lead the way with fate mapping?
Why?
Chick/quail studies
- Quail and chick embryos are very similar - develop at the same pace/size
- BUT, there are antibodies that particularly recognise the surface of the nucleus of the QUAIL embryo but NOT the CHICK embyro
- Can visualise the quail cells in chick host
How do you produce a fate map using quail/chick studies?
- Transplant quail tissues into chick in a specific area
- Let the chick develop
- Later on, use immunohistochemisty to find the position of the tissues of the quail
- Do this many times - produce a fate map of the early chick embryo
What is the problem with using quail/chick studies to produce fate maps?
Cannot get single cell resolution
What is the brainbow technique?
Label cells by introducing a fluorescent construct into the animal
What is the process of the brainbow technique?
- Create a transgenic reporter but introduce MANY genes encoding for DIFFERENT FLUROPHORES
- Each flurophore - framed by a DIFFERENT PAIR of loxP sites that are slightly different from eachother
- Different loxP sites are recognised by different cre recombinases (cell specific?)
- Can only have ONE TYPE of excision - Which excision determines which gene is left
- When cross the animal with cre recombinase - give a WIDE RANGE of colours that are distinct in each cell type where recombination has taken place
What can the brainbow technique be used for?
- To follow the descendants of cells (fate mapping)
What is an advantage of the brainbow technique?
Good precision
What animal models can the brainbow technique be used in?
Mice
Zebrafish
