Animal Development (excluding development of Xenopus, Drosophila and Mouse) Flashcards
What makes a good animal model?
any species that’s been widely studied, easy to maintain, easy to breed, low cost, known genome sequence, experimental advantages.
What would make an animal model good for genetics studies?
- large array of mutants
- large no. of offspring
- short generation time
What would make an animal good for embryology?
- robust embryos
- easily manipulated
- large no. of embryos
- external development
What would make an animal good for genomics?
- relevance to human genome
- disease model
- drug testing
Examples of developmental models (invertebrates)
- CAENORHABDITIS ELEGANS - worm
- DROSOPHILIA MELANOGASTER - fly
Examples of Developmental models (vertebrates)
- DANIO RERIO - zebrafish
- XENOPUS LAEVIS - African clawed frog
- GALLUS GALLUS DOMESTICUS - chicken
- MUS MUSCULUS - mouse
Definition of Blastula stage
ball of cells
Definition of Gastrulation
cell movement producing germ layers
Definition of germ layers
ectoderm, mesoderm, endoderm
Definition of neural plate stage
embryo after gastrulation
Definition of neurulation
cells move to roll up neural tube
Definition of dorsal and ventral
back and belly
Steps of animal cloning (somatic cell nuclear transfer)
1) UV radiation of unfertilised egg
2) nucleus of cell of blastula taken and injected into enucleated egg
3) albino frog
Steps of cloning with differentiated adult cells
1) Cultured adult skin cells
2) Uv radiation destroys DNA in nucleus of egg
3) removal of nucleus from cultured cells
4) transfer of nucleus to egg
5) tadpole develops
What are transcription factor codes in development?
- bind to DNA to cause transcription of genes, lots can be active at same time
- If diff transcription factors activated later on in development, it may differentiate differently and differ from wild type.
Wild type of is blank / TF1 / TF1 + 2
What are inductive signals and what is their role in development?
- signal transduction carries signal from membrane to nucleus
- cause activation of diff TF
- signalling cell manufactures inductive signals
- further away you get lower conc of IS
- receptors span plasma membrane, signal binds, protein enters nucleus and activates trans factor
What dose of Inductive signal does TF1 and TF2 need?
- TF1 is medium
- TF2 is high
- low dose = no TF switched on
What is experimental embryology (using signal cells)
- taking and moving signal cells to other end of cells to see what would happen , high dose at each end
Why are amphibians well suited to experimental embryology? (stage 1 of understanding inductive signals)
- mother produces lots of eggs
- embryos develop externally
- large embryos
- tissue heals well
- embryos wont die if taken elsewhere
What did Hans Spearmann do in his experiment and what were the aims?
- carried out experiment in Newts in 1900’s
- took fertilised eggs, wanted to split in half but not separate them
- wrapped hair around an egg to keep cytoplasm on one side and nucleus on other
- one cell would go to the other side so he got two early embryos occurring
- only one side had grey crescent, other side develops oddly
What occurs in a Spearmann Organiser Graft?
- Gastrala stage embryo –> cut out a piece of tissue at dorsal region where dorsal lip would occur
- transferred to ventral half of gastrin stage embryo
- allowed embryos to develop
- got two headed twins
- THEREFORE Spearmann Organiser Graft produces all signals you need to induce second axis
What is an inductive signal?
Extracellular signal which causes cells to follow different fates.
What is cell fate?
- what cells will end up being due to differentation
What changes in fate does a Spearmann Organiser Graft make?
- induces dorsal fate in cells which would otherwise follow ventral
- cells in developing embryo that induces development or central nervous system
What is the role of a signal transduction?
- Relays the message from the extracellular signal from the membrane to the nucleus where it activate gene expression e.g Transcription Factor
What occurs in stage 2 (Generating Mutants) of understanding inductive signals?
- make plasmid library containing 1000’s of random genes
- use a needle to inject mRNA into embryo
- lots of protein translated by embryo
- abnormal embryo / tadpole due to overexpression
What happens if fertilised egg treated with UV radiation?
- forms into belly piece embryos
- so lots of diff mRNA’s injected to find one with resolves the head development
What does noggin do?
- promote dorsal structures
- it’s a secreted signalling molecule
- it’s expressed in the Spearmann Organiser - cells far away follow ventral fate, cells close follow dorsal fate
- doesn’t turn on the HOX gene
What occurs in stage 3 (checking whether gene is expressed) In-situ hybridisation of understanding inductive signals?
1) Add RNA probe attached to tag
2) Add enzyme which recognises tag, enzymes are attached to antibodies
3) Add substrate to solution to form a coloured precipitate
What is superficial cleavage in Drosophila?
- divides as nuclei not cells, goes from one nuclei to 2 to 4 etc.
- nuclei increase
- after 9th division nuclei moves to outside edges of zygote, called syncytial blastoderm
- pole cells move to end of zygote
What is a genetic screen and what are the stages to it?
- technique used to identify and study an interesting phenotype within a mutated population.
1) Generate the mutants
2) Identify gene that has mutated
3) Find where genes expressed using forward genetics
What occurs in stage one of a genetic screen (generating the mutants)?
- F1 generation leads to recessive phenotype, not clear which individuals have mutated gene though
- so gene crossed with wild type to make F2 generation
- F2 then inbred and from this you can see which families contain the mutated gene (they will have inherited 2 copies)
What are homeotic mutants?
- expressed in diff anterior/posterior positions
What are homeotic genes?
- Homeobox transcription factors
- activate/repress genes required to specify organs/cell types
- control homeobox domain
- control pattern of body formation
What is collinearity?
- order of expression in embryo matches order of genes
Drosophila MOST OF HEAD (position in body related to homeotic gene wildtype)
lab and Dfd give antennae
Drosophila Thoracic seg 1 (position in body related to homeotic gene wildtype)
scr gives pair of legs
Drosophila Thoracic seg 2 (position in body related to homeotic gene wildtype)
Antp gives pair of legs and wings
Drosophila Thoracic seg 3 (position in body related to homeotic gene wildtype)
Ubx and Antp give pair of legs and halteres
Drosophila Abdominal seg 1-4 (position in body related to homeotic gene wildtype)
abdA and Ubx and Antp give no outgrowths
What are the homeobox genes like found in mammals compared to Drosophila?
- 38 hox genes, similar
What does lineage restriction mean?
cell will only give rise to certain cell types
Issues with doing a genetic screen on a mouse
need something cheaper smaller and easier to breed
What is reverse genetics?
add/manipulate gene then look at phenotype
- permanent change
Transgenics in a mouse (overexpressing gene product)
- male pronuclei larger
- use needle to inject DNA into male pronuclei
- use fungi plasmifs in bacteria (replicate), use promotors in a test tube and DNA polymerase to drive transcription –> lots of copies of gene
- DNA will intergrate into chromosomes
- done in dish, inject eggs into mother
What is a targeting construct?
- knock out genes
- made up of DNA stitched together, sequences contained in plasmids
- purified from bacterial plasmids, linearized and used within ES to cause genetic deletion
- DNA construct moves into ES nucleus, recombination
- gene of interest removed and replaced with a marker and reporter
- ES cells reseeded into culture dish- grow
What is a chimeric mouse?
- combo of normal and targeted cells
Microinjection of ES cells into host blastocyst
- take blastocyst embryos, inject into cavity of blastocyst and expel out the ES cells
- get recombinant blastocysts
- transplant into uterine horn not oviduct of surrogate mother
