How can we study development?
- Anatomically (descriptive embryology, normal morphology and histology, fate mapping)
- Experimentally (commitment and induction- e.g. needle experiment/chopping stuff up)
- Genetic (more commong now- identification of genes controlling development and gene interactions by the manipulation of genes)
Name desirable characteristics for studying development?
- Relevance/representative
- Accessibiltiy/availabiltiy
- Experimental manipulation
- Genetics
- Cost/space
Descrive how descriptive anatomy can be studied
- light/electron microscopy
- histological sections
- morphological and anatomical description of how embryos change over time
- basis for understanding, but does not usually follow cell movement/origins
- no mechanistic information
Describe what fate mapping and lineage analysis is
- Following cells to see where they end up (fate)
- Defines patterns of cell migration
- Defines origins of cells in formed structures
e.g. label a cell population–> graft into animal–> see genotype expressed as a phenotype from A to B and the pathway inbetween
Explain in-situ hybridisation
- Look at mRNA expression
- Make a probe in the lab complementary to mRNA you wish to identify
- Add this to the embryo
- Wash the probe off
- Incubate embryo and recognise DIG (DIG= modifed epitope with tag)
- e.g. DIG binds to AP-conjugated anti-DIG- binding causes substrate to release coloured ppt
Describe immunostaining
Detect protein expression- amplify signal and include tissue in
- add primary Ab
- add secondary Ab (diff species to primary)
e. g. Brachyury (mesoderm marker in mouse gastrulation)
Describe subtractive hybridisation
- Not so common any more though lol
- identifies differences in gene expression between regions of the embryo OR identify differences in gene expression between normal and mutant embryos
- isolate the mRNA from the two samples make cDNA and modify one sample e.g. biotin
- hybridise two samples
- remove the genes common to both samples (e.g. using streptavidin)
- isolate remaining genes
Desrive microarray experiment
- RNA isolation of sample A and B
- cDNA generation
- Label probe- reverse transcription with fluoroescent tags (both samples with 2 different colours)
- Hybridise to array (glass chips with sports of short DNA)
- image to see if Sample A > B, B >A, A=B
Advantages/disadvantages of studying mice:
- develops in utero
- genetics- has lots of background info
- can be manipulated (transgenics)
- mammal therefore closely related
- 21 day gestation is short = good
- expensive to breed/store
- ethical/legal concerns with testing on vertebrates
Ad/Disad of frog (xenopus laevis)
- large embryos= easier to manipulate
- develops in simple salt solution/explants
- rapid development (~30 hrs to hatch)
- poor genetics (X. tropicalis better than X. laevis)
- readily injected with RNA, labels etc.
Ad/Dis of drosophilia
- not transparent therfore difficult to see inside
- short embryogenesis (~24 hours)
- Easily bred and maintained (14 day generation time)
- Genetics- well understood; easy to manipulate
- Easy to form mutants
Describe the drosophila cuticle patterns
- Drosophila larva has 14 unique segments
- each segment has a specific pattern of denticles
- each segment has polarity
How is the drosophila body plan established?
- maternal genes are expressed in gradients at the anterior and posterior poles
- gap genes are activated and in turn, established pair-trule gene (eve, ftz) pattern
- Pair-rule genes establisg polarity genes (engrailed, wingless)
- Segment identity is establisged at cellularisation by competing Tfs
- maternal biocoid activates hunchback
- gap genes inhibit eachother and establisg the position of pair-rule gene expression
Ad/disad of studying C. elegans
- transparent
- short embryogenesis (~15 hrs)
- invariant lineage
- Genetics. manipulation
- 558 cells at hatching- origin of cell is known
Ad/disad of studying zebrafish (danio rerio)
- rapid development (<24 hr)
- genetics- well known
- mutants- easy to make
- transparent embryo
- short generation time (3 months)
Ad/disad of studying gallus gallus (chick)
- accessible embryo; can incubate live embryos
- experimental manipulation achievable
- poor genetics
- embryogenesis ~4 days (21 days to hatch)
What causes ecotopic eye induction in drosophila?
What happens if this is mutated?
- Pax6= ectopic eye induction
- Ectopic eyeless (or mouse Pax6)- small eye- mutation in Pax6
How can you identify genes controlling development?
- genetic analysis: mutagenesis screens in drosophila, xenopus mouse and zebrafish (forward genetics)/reverse genetics- mutation of specific DNA sequences and analysing phenotype/transgenics
- molecular methods: methods to identify genes with restricted expression patterns in the embryo
Describe mutagenesis screens
Saturating mutagenesis screens in Drosophila and identification of vertebrate homologues of mutated genes
similar screens in zebrafish
many mouse mutations exist from smaller screens and spontaneous events but more recently, large-scale screens have been undertaken
Describe genetic screens for recessive mutations in zebrafish
- ENU (ethyl nitroso urea) mutagenesis
- Keep F1 males
- Cross F2 animals
- 1 in 16 is homozygous for mutation
- Establish line from F1
- Identify genes by -complementation anlysis, genetic mapping, positional cloning
What happens when you study the interactions of genes controlling development?
- determining the exact expression domains within the embryo: in-situ hybridisation to detect RNA, immunohistochemistry to detect protein
- genetic analysis of interactions (cross mutants with similar phenotypes to see interactions)
- Misexpression gain and loss of function studies in vitro and in vivo
Describe mix-expression studies
- express a gene in a region of the embryo (or at a time) where it is normally not expressed
- study the effect on development and/or expression of other genes
Descrive gain and loss of function
- For secreted factors, use protein on beads or cells constitutively expression protein applied to regions of the embryo
OR apply neutralising antibodies in same way- for cell surface proteins
-For IC targets, can use retrovirus with gene under suitable regulation injected into cells (sometimes together with marker)
-Small synthetic RNA of derivatives (morpholinos) single strand oligomers or oligos complementary to mRNA sequence. Inhibit protein synthesis or splicing RNAi to degrade RNA targets
Altering protein function to generate loss/gain of function mutants:
- TFs bind to specific regulatory sequences to control gene expression
How does gene inactivation by CRISPR-Cas9 occur?
Clustered Interspersed Short Palindromic Repeats
Based on bacterial adaptive immune system
Developed in 2013 and rapidly adopted since then
Can be used for a wide variety of species and cells in culture
Requires a guide RNA with a Protospacer Adjacent Motif (NGG for Cas9)
sgRNA (single guide) made–> add to Cas9 protein–> this cleaves specegic target –> cellular error-pone repair “knocks out” gene
