Week 2

Question 1

What is anatomical embryology?

Answer 1

The study of how anatomy changes within and between embryos

Question 2

What is genetic embryology?

Answer 2

How genes control development

Question 3

What is experimental embryology?

Answer 3

The mechanics of development

Question 4

What are the pros of model organisms?

Answer 4

Easy to breed all year round

Easy to maintain in a lab

Easy to keep large numbers

Fast development

All have a sequenced genome

Display similarities to humans

Question 5

What are the cons of model organisms?

Answer 5

No single model is sufficient
- Most research uses a combination

Other considerations: Suitability to answer your research question, cost, availability

Question 6

Why are model organisms used?

Answer 6

Experimental and anatomical embryology easier in larger embryos that develop externally

Genetic embryology easier in small diploid animals with short generation times and many offspring

Many biological processes are the same in all animals - particularly during development

Question 7

Describe why fruit flies are used as animal models:

Answer 7

Drosophila melanogaster

• Short lifespan
• Easy to handle large numbers
• Easily mutated (diploid)
• Ideal genetic model
• However, not vertebrate

Question 8

What is really good about drosophila?

Answer 8

44% (6057/13600) of fly genes show homology with human genes

62-75% of human disease appear to be conserved in the fly

An ability for reverse genetics as well as forward genetic screens to saturation

Full annotated sequence of melanogaster genome as well as 11 other Drosophila genomes

Question 9

Why are zebrafish used as animal models?

Answer 9

Danio rerio

• Small, cheap, easy to keep
• Transparent - good for anatomical development
• Fast development
• Amenable to genetic embryology
• Somewhat amenable to experimental embryology
• Easy to mutate

Question 10

How can you alter the genetics or gene expression of an animal?

Answer 10

Mutagen

Antisense RNAi

Antisense morpholino oligonucleotide

Question 11

What is the action and longevity of mutagen in altering the genetics or gene expression of an animal? What organism can it affect?

Answer 11

Changes genetic code: can be loss-, gain- or change-of-function

Longevity: inherited/permanent; Transient

Organism: Zebrafish, Drosophila, C.elegans, yeast. Canopus and mice to a lesser extent

Question 12

What is the action and longevity of Antisense RNAi in altering the genetics or gene expression of an animal? What organism can it affect?

Answer 12

Degrades specific mRNA: Knockdown

Longevity: Transient or permanent

Organism: Drosophila, Xenopus, cell culture. Chick? Yeast?

Question 13

What is the action and longevity of antisense morpholino oligonucleotide in altering the genetics or gene expression of an animal? What organism can it affect?

Answer 13

Inhibits mRNA splicing or translation: Knockdown

Longevity: Transient

Organism: Zebrafish, Xenopus, Cell culture, Chick?

Question 14

What are mutagens?

Answer 14

Traditionally used in mutagenesis screens

In a screen you want to mutate every gene possible so you can learn what the mutant phenotype is for each gene

Can do this in a reverse or forward way

Question 15

What is reverse mutagenesis?

Answer 15

Mutate at random > view phenotypes > identify mutated gene

Question 16

What is forward mutagenesis?

Answer 16

Identify a gene > mutate that gene > view phenotype

Or

Mutate at random > Identify animals with mutation in specific gene > view phenotype

Question 17

What are mutagenic agents?

Answer 17

Chemicals such as EMS or ENU that intercalate into DNA

Transposons that integrate into the genome (P-elements in Drosophila, Tol2 or sleeping beauty in Zebrafish)

Retroviruses that integrate into the genome

Genetically engineered agents of gene editing such as CRISPR, TALENS and Zinc fingers

Question 18

What do we see in an EMS mutagenesis screen in Drosophila?

Answer 18

Balancer x EMS

25% Balancer homozygotes: die as larvae

50% Balanced mutant stock: viable - Breed these to keep the line

25% Homozygous for mutagenised chromosome: Screen embryos for phenotype

Similar in zebrafish but no balancer chromosomes

Question 19

What happens in CRISPR?

Answer 19

Design a guide RNA

Inject guide RNA and capped mRNA encoding Cas9 into the embryo

The Cas9 is translated

The Cas9 causes double-strand breaks in the target DNA

Breaks are repaired, sometimes incorrectly

Effects are transient unless the germline is affected and you breed the mutation on

Question 20

What is a loss of function mutation?

Answer 20

If you generate a loss-of-function for a gene/protein, the resulting phenotype tells you what the normal (now lost) function of the gene/protein is as this is the function that has changed. The mutations are often recessive.

E.g. A premature stop codon that means the mRNA undergoes nonsense mediated decay, so no protein is made at all

E.g. Change of an amino acid required for catalytic activity of a protein (assuming no other functions elsewhere in the protein)

Question 21

What is a gain or change of function mutation?

Answer 21

If you generate a gain- or change-of-function for a gene/protein, the resulting phenotype tells you what happens if a gene/protein is expressed at a higher level, in the wrong place, or if the protein function is changed. These mutations are often dominant

E.g. a mutation in a drosophila homeobox gene which causes ectopic expression

Question 22

What do we know about Ultrabithorax mutant (Ubx/+; Homeotic gene)?

Answer 22

• T3 converted to T2
• Ubx no longer expressed in T3
• Antp expression expands from T2 into T3

4 wings instead of 2!!

Question 23

What do we know about antisense RNAi (in drosophila)?

Answer 23

RNAi uses ds RNA that is processed by the Dicer ribonuclease in the embryos to produce short interfering 21-25 nucleotide RNAs (siRNA)

siRNAs guides sequence specific degradation of mRNA leading to post-transcriptional silencing of the target locus

dsRNA needs to be complementary to the locus you want to knock down

injection of RNAi causes a transient effect

You can introduce RNAi as a transgene and control its expression using the Gal4-UAS system

Question 24

What is the mechanism for endogenous antisense RNAi?

Answer 24

pri-miRNA <— Genomic DNA

Nucleus:
- Drosha
- DGCR8

Exp5

Cytoplasm:
- Dicer

Mature miRNA

RISC loading

RISC Target transcript

Target degradation or repression

This mechanism is harnessed for genetically engineered RNAi to target chosen mRNAs

Question 25

What do we know about antisense morpholino in Zebrafish?

Answer 25

A morpholino is a 25mer with a nucleic acid base, a morpholine ring and a non-ionic phosphorodiamidate intersubunit linkage. Morpholinos act via an RNAse H-independent steric blocking mechanism Their effect is transient

Question 26

What do we design the morpholino to be complementary to?

Answer 26

The promoter region of the chosen gene to inhibit the translation of that gene. This will stop the translation of maternal mRNA deposited in the egg plus zygotically expressed mRNA A splice site in the chosen gene to inhibit splicing of transcripts from that gene. This will only affect zygotic transcripts as maternally deposited transcripts are already spliced

Question 27

How do you confirm the cause of the phenotype?

Answer 27

You designed an experiment to have an effect, but has it turned out as designed? - Off target effects - Toxicity You have performed random mutagenesis, but is the identified mutation really the cause of the phenotype? - A mutation in another gene might cause the phenotype

Question 28

How do we identify off-target effects and toxicity for RNAi (drosophila)?

Answer 28

Loss of target protein (western blot) Loss of target mRNA (RT PCR) Similar phenotype to a mutant for the same gene Rescue of phenotype after injection of mRNA of the equivalent gene from a related species gene (can't have the RNAi target sequence in it)

Question 29

Identifying off-target effects and toxicity for a morpholino (zebrafish)

Answer 29

Loss of protein (western blot) if morpholino is directed at the promoter Loss of normal splicing (RT PCR) if morpholino is directed at the splice site Loss of a fluorescent reporter that has a morpholino binding site Similar phenotype to a mutant in the same gene Similar phenotype to another morpholino for the same gene Rescue of phenotype after injection of mRNA of the same gene (can't have the morpholino target sequence in it) Cell death phenotype still occurs when p53 is also knocked down (p53 is required for cell death due to toxicity)

Question 30

How do we confirm that a mutation causes a phenotype?

Answer 30

A closely linked mutation/insertion might be the cause of the phenotype, not the mutation you have identified: - Rescue an embryonic phenotype with injected mRNA of the wild type gene - Use morpholino (fish) or RNAi (fly) knockdown to phenocopy the mutant - Other alleles should have a mutation in the same gene

Question 31

How can we avoid early phenotypes in Zebrafish?

Answer 31

By injecting mRNA Making late zygotic oep-/- zebrafish injecting oep mRNA

Question 32

What does injecting oep mRNA do?

Answer 32

Produces late zygotic oep-/- zebrafish Enables study of development of the asymmetric brain

Question 33

How do we avoid early phenotypes in drosophila?

Answer 33

By creating genetic mosaics Homozygous mutations may cause lethality before the process you are interested has begun. You can study homozygous mutant tissue using mosaic animals The FLP FRT system After induction of FLP they bind to FRT and recombination occur Then mitosis Then mitotic clone (homozygous mutant cell) and cell with two copies of the wild type gene are produced

Question 34

How do we avoid early phenotypes in zebrafish pt 2?

Answer 34

By creating genetic mosaics by transplantation Donor embryo --> Acceptor embryo

Question 35

What are some other genetic techniques for avoiding early phenotypes in both drosophila and zebrafish?

Answer 35

Transposons can be engineered To express fluorescent markers with expression like endogenous genes To express a functional zebrafish gene (simply, and also using the Gal4-UAS system) To contain a STOP signal so stops translation of genes it is inserted into

Question 36

What do we know about the Zebrafish Gal4-UAS system (also in drosophila)

Answer 36

Microinjection of plasmid DNA with a GAL4 trap construct and transposase mRNA Injected fish bred with reporter fish Produces GAL4 expression and reporter expression The synthetic transposase mRNA and a transposon donor plasmid containing a Gal4 trap construct are coinjected into zebrafish fertilised eggs. The trap construct is excised from the donor plasmid and integrated in the genome. Tol2 insertions created in the germ cells are transmitted to the next generation. In the double transgenic embryos obtained from a cross of the injected fish and the UAS-reporter fish, Gal4 expressing cells are visualised by the fluorescent reporter

Question 37

What are modifier screens in drosophila?

Answer 37

In order to find genetic interactions, you look for mutations that affect the phenotype of another mutation Only done in genetic organisms where it is easy to keep thousands of strains, such as fruit flies (not often in zebrafish)

Question 38

How do you do a modifier screen?

Answer 38

Either conduct a mutagenesis screen in the background of an existing mutation of interest Cross in lots of known mutations or transgenes to the existing mutation of interest e.g. crossing the deletion library into the background of flies over-expressing Cln3 in the retina

Question 39

What are the advantages of using zebrafish for drug and toxicity screening?

Answer 39

Just add drugs to the water High-throughput screening strategies - many drugs on many animals Many diseases have visible phenotypes, especially as zebrafish are transparent Zebrafish anatomy and behaviour is simpler to examine Mutant + Drugs or small molecules --> Phenotypic rescue + toxicity --> Therapeutic agent --> Mammalian trials

Question 40

What are the concerns/disadvantages of using Zebrafish for drug and toxicity screening?

Answer 40

Drug must be water soluble Need a phenotype that can be screened automatically Biology may be different from mouse or human Drug response may be different Blood brain barrier develops at a different age - some drugs might rescue a fish as it crosses blood brain barrier, but not mice or humans

Question 41

What phenotypes can be screened automatically?

Answer 41

Fluorescent reporter Behaviour

Question 42

What are some well known drosophila genetic screens?

Answer 42

EMS mutagenesis - Mutations affecting segment number and polarity in Drosophila P-element screen - Enhancer trap screen-Beta galactosidase expression controlled by endogenous promoter - P-element insertion causes mutation - Berkeley Drosophila genome project gene disruption project. Single P-element insertions mutating 25% of vital Drosophila gene - P-element transposon jumped around the genome by supplying transposase - P-element insertion causes mutation Subsequent P element and mutagenesis screens - Subsequent screens using in situ hybridisation or immunohistochemistry to visualise internal structures Modifier screens FLP/FRT mosaic germline screen - Mutations introduced into flies with FRT sites and FLP under heat shock control - When heat-shocked, FLP induces recombination at FRT sites and any mutations further from centrosome become homozygous in clones of cells RNAi screen

Question 43

What are some well known Zebrafish genetic screens?

Answer 43

Large-scale zebrafish ENU mutagenesis screen for developmental defects - Assay - visible phenotypes and retino-tectal projection - Recovered 4264 mutants Large-scale zebrafish ENU mutagenesis screen for developmental defects - Assay - visible phenotypes - Recovered 695 mutants Large-scale retroviral insertional mutagenesis screen for developmental defects - Assay - visible phenotypes - Several papers published Large-scale zebrafish ENU mutagenesis screen for developmental defects - Assay - visible phenotypes, in situ hybridisation, whole-mount immunohistochemistry Large-scale zebrafish ENU mutagenesis screen for post-embryonic phenotypes - Assay - visible phenotypes, in situ hybridisation, whole mount immunohistochemistry Antisense morpholino screen - Reverse genetic screening reveals poor correlation between Morpholino-induced and mutant phenotypes in zebrafish Crispant screen

Question 44

Why do we need animal models?

Answer 44

Useful for looking at normal processes in development Useful for looking at disease processes

Question 45

What are some commonly used animal models and their advantages?

Answer 45

Fly (drosophila) Fish (Danio) Mouse All have relatively short lifespans 2-2.5 years for mice/fish Genetic models are available (gene knockins/knockouts)

Question 46

What are some less common animal models and why should we use these rather than one of the more common ones?

Answer 46

Dog Chicken Newt Pig In some situations these models are better/more appropriate for answering specific questions

Question 47

What do we use to investigate normal embryonic development?

Answer 47

Whilst the mouse can be used to answer many questions in embryology through the use of transgenics, the chick embryo is still a widely used model in embryology This is because the chick model has several advantages over using mice

Question 48

How accessible is the chick embryo?

Answer 48

As eggs develop external to the mother (unlike mice and most other mammals) embryos are easily accessible and can therefore easily be manipulated surgically to examine tissue/tissue interactions, cell fate etc This is particularly useful in avians as it is possible to do interspecies tissue transplants to follow the fate interactions of specific tissues As there are antibodies that specifically recognise quail cells (but not chicken), quail cells transplanted into a developing chick embryo can be discriminated

Question 49

What specific questions can we ask by using quail cells in chick embryos?

Answer 49

e.g. which cells of the embryo give rise to axial tendons? This can be answered easily using somite transplantation techniques --> Transplanting a quail embryo somite into a chick embryo

Question 50

Why is using chick embryos a better alternative to mouse embryos?

Answer 50

With mouse genetic models, development of new transgenics can be time consuming and costly Chick embryos offer a cheaper faster alternative to this as they can be genetically manipulated using retroviral technology

Question 51

How can specific tissues of the embryo be infected?

Answer 51

Using localised microinjection of virus This can be used to overexpress a protein of interest in a particular tissue or Downregulate expression of endogenous protein using RNAi producing virus

Question 52

What animal models do studies on regeneration use?

Answer 52

Non-standard models You can use the mouse as a model of regeneration, but its capacity, and therefore its usefulness, are limited.

Question 53

What do we know about regeneration in embryonic/early postnatal mice?

Answer 53

Msx1 is expressed in the tips of the embryonic postnatal digits Digit tips regenerate when amputates within the Msx1 expressing region

Question 54

What model organism should be used to investigate the fundamental mechanisms that underlie regeneration?

Answer 54

It is essential to study an organism with extensive regenerative capacity For example - Urodele amphibians (newts/salamanders)

Question 55

What happens in urodele tissues in response to injury?

Answer 55

A healing (scarring) response is not elicited following injury, but rather a regenerative response Differentiated tissue revert back to a non-diferentiated mesenchymal state, proliferate and then re-differentiate to give rise to the new structures of the regenerate

Question 56

What has using a urodele regenerative model taught us?

Answer 56

It has been possible to determine some of the factors that allow for the production of a regenerate e.g. Retinoic acid and nAG (anterior gradient protein) produced by Schwann cells And also what gives the regenerating tissues positional identity e.g. Retinoic acid, Meis - Control GFP transfected cells remain in the distal/mid part of the limb - Meis and GFP transfected cells move to the proximal part of the limb

Question 57

What is Duchenne Muscular Dystrophy?

Answer 57

A childhood onset disease that results in muscle weakness, wasting and eventually death in humans The disease is known to be due to a loss of the protein dystrophin A protein that connects the actin cytoskeleton to the sarcolemma in muscle

Question 58

What do we know about Duchenne Muscular Dystrophy in other model organisms?

Answer 58

Mouse DMD is biochemically the same as humans but with lesser clinical manifestations Dogs have severe clinical manifestations

Question 59

What can exon skipping do?

Answer 59

Re-establish the expression of dystrophin resulting in partial functional recovery in DMD golden retrievers

Question 60

What does Huntington's disease do?

Answer 60

Affects the brain's basal ganglia

Question 61

What do we know about modelling huntingtons disease in a mouse?

Answer 61

Can be modelled in mouse but does not fully recapitulate the human disease Characteristic apoptosis of neurons absent in mice The physiology, structure and development of the pig brain is much closer to human so this may be a better model - Increased apoptosis, more similar to human cases, seen in transgenic pig

Question 62

Does similarity of physiology and structure mean a better animal model?

Answer 62

Not always For example, in pig models of alzheimers disease, overexpression of beta amyloid protein does not result in dementia Therefore it is key that you choose your model such that it will be appropriate for what you are testing