What is expression?
Presence/absense of protein or mRNA
What are techniques to measure expression?
Immunostaining of whole embryo (protein), Western blot (protein), in situ hybridization (mRNA), Northern blot (mRNA), QRT-PCR
What does immunostaining identify, and what does it give no info about?
It identifies time and cell/tissue (space), but gives no info about function
What info does a Western blot provide?
What does each well of a western blot correspond to?
A tissue type
In situ hybridization can only be applied to _____ tissue sections and embryos.
What labels are used in in situ hybridization, and does one have an advantage?
FITC (green fluouresence) or DIG (digoxygenin); DIG can amplify to show low-level expression, but once used, it's done.
Why look for RNA as opposed to protein?
Regulation can occur before translation; or there can be a delay in translation.
What does a Northern blot measure?
mRNA levels, temporally and spatially
What kind of probe is used in a northern blot?
radioactive, fluourescent, etc.
What is a weakness of a Northern blot?
It requires a lot of embryonic tissue
What is needed for QRT-PCR?
About 10 embryos, a solution which dissolves and homogenizes, PCR primers, RT primers for cDNA from mRNA, probe
What is PCR done on in QRT-PCR?
What is a disadvantage of QRT-PCR?
It's very expensive
What are techniques to identify cell lineage?
Stem cell tracer, cell ablation, fate maps,
How does lineage tracing with stem cells work?
Inject a stem cell with a fluourescent tracer and observe progeny.
What can cell ablation tell you?
What are progeny; what are necessary; info on cell-cell interaction
What is a restriction of cell ablation?
It does not work on mammals.
What is an inducible promoter?
cell-specific; toxin only released at certain point in development;
What are micromeres?
early cell (wrt zygote)
What does a fate map show?
relative proportion and arrangement of cells; migration of cells; contribution to later development
What is a fate map?
A graphical representation of what early embryonic cells will give rise to later during development or in the adult.
What animal is fate mapping easiest in?
What animals does transplantation work in?
Frogs and chicks
What constitutes a negative experiment with respect to transplantation?
Removing cells to see what migrates or what is missing.
Why transplant between different species?
The differences are more clear-cut.
What is a disadvantage of doing a transplant?
The cellular mechanisms responsible for inducing the change is unknown.
When are cells removed in a transplant experiment?
In a "rescue" experiment. Otherwise, the cells are simply added to the animal.
Name the different terms for describing body plans in animals.
rostro/anterior, animal/top, posterior/caudal, bottom/vegetal, dorsal/back, ventral/stomach
Describe an explant.
From a frog, take a chunk of tissue/embryo and grow it in culture. Observe development: somite formation, neural tube formation, morphological change, etc. It is a non-genetic experiment.
Why do an explant?
It eliminates potential interaction with other cells; easier to visualize.
What is a limitation of an explant?
Context is missing, so results can be difficult to explain.
What is a confocal microscope?
It allows for "optical" sections of a specimen to be observed without actually physically sectioning the specimen. Results often need to be verified with explants.
What are the genetic model organisms?
- Fruit flies
- C. elegans
- Thale cress
What are four non-genetic model organisms used in development?
- sea urchin
What makes a genetic model organism?
- controlled genetic crosses
- transgenic tools
- system to eliminate/inactivate/introduce a gene
- ability to screen for mutants
Why isn't a frog a genetic model organism?
Why isn't a leech a GMO?
- no transgenic tools
- No way to screen for mutants
- no way to induce mutations
Why isn't a chick a GMO?
- No transgenic tools
- Difficult to mate
- can't screen
Why isn't a sea urchin a GMO?
- no techniques for inducing mutations
- can't screen
List 5 molecular techniques
- Producing mutants
- In situ hybridization
- Knockdown (RNAi and morpholinos)
How do you determine function with a genetic technique?
You must remove genetic component to verify function.
- transient (temporary; short-lived)
- Prevents translation, i.e. "removes" protein
How does knockdown work with RNAi?
A plasmid with a short complimentary sequence and a hairpin loop binds to the mRNA and attracts enzymes which cut out the RNA sequence of interest. It can be effective for up to two weeks.
How does knockdown work with a lentivirus?
Plasmid is integrated into the cell chromosome, so this knockdown effect can be passed on to later generations.
How would you determine if knockdown worked?
How do you determine sufficiency?
Ectopically apply the protein or mRNA. For mRNA, a 5' cap is needed. Immunostaining would need to be performed to make sure the mRNA is present.
How do morpholinos achieve knockdown?
They bind directly to the mRNA preventing translation.
What are a couple of genetic techniques?
Knock-in and knockout
Overproduce a protein. Look at threshold events. Look at specific cells/tissues. It is a targetted insertion.
A mutation/deletion of a gene results in no mRNA and thus no protein.
What is required to do create a knockout animal?
A plasmid/vector with the (broken) gene of interest and enough flanking DNA to maximize the chance of RECOMBINATION.
Why might knockdown fail?
- Redundancy - multiple genes doing the same thing.
- Recombination didn't occur.
How would you tell if knockdown worked in a mouse?
Take a tail sample and do PCR on original sequence/region. Must be done after first litter.
What are advantages of studying development in the leech?
- Genome sequenced
- Large embryo that are easily manipulated (e.g. morpholino knockdown) and can follow progeny
- Inexpensively bred in labs - simple salt solutions
- Early lineages mapped out - know how things should develop, so deviations are easily recognized
- Bilateral symmetry
- Few cells: Teloblasts/stem cells well-studied
- Development is conserved
What are some disadvantages of studying leeches?
- Not a genetic model
What are a few common cellular techniques used in leeches?
- Cell ablations
- In situ hybridizations
What is a common molecular technique used in leeches?
Describe cell division in leeches.
Spiral cleavage (stereotypic cleavage patterns), i.e. unequal cell division, takes place. Daughter cells divide; not stacked on top of each other, but in a spiral.
What effect does this have on mitosis?
Different sized cells means that spindles are not symmetrically placed/formed.
When does meiosis occur in a leech embryo?
When the embryo is "extruded" from the "mother"
What gives rise to the unsegmented portion of leeches?
What gives rise to the segmented portions?
What is gastrulation?
The formation of germ layers: ectoderm (outside and CNS), misoderm, endoderm (gut)
What is the teloplasm?
Teloplasm is the yolk-free cytoplasm of the early zygote. Maternal mRNAs localize the teloplasm towards the cortex prior to the first cleavage.
What are some advantages of studying the fruit fly?
- Easily bred
- Easy to mutagenize
- Fast life cycle
- Sequenced genome
- Conserved functions for some genes in vertebrates
What are some different types of homologies?
- DNA - similar sequence, but not necessarily similar function
- Biochemical - transcription factor might have same chemical/physical action, but maybe not same function/result
- Biological - same function, e.g. engrailed: brain development
What are parasegments?
In the fly embryo, these structures look like "clumps", and are precursors to segments in the adult
What is one major difference between the development of a leech embryo and a fly embryo?
The fly embryo is one big multinucleated cell, i.e. cyncytium. Nuclei turn off/on depending on the location. Nuclei migrate to the cell periphery where cytokinesis begins.
Describe axis determination in flies.
- mRNA from mother is put into the egg.
- Endoderm (gut): posterior and anterior
- Mesoderm: ventral
- Ectoderm: dorsal, and along sides
Describe the results of early screens in fruit flies.
- Egg polarity genes: (maternal) established the AP and DV axis
- Gap genes: broad regions along the AP axis (normal development)
- Segment polarity genes: organize the AP pattern in each segment
- Selector genes: determine segment identity
What 4 genes regulate the A/P axis in flies?
- Bicoid: localized
- Nanos: localized
- Hunchback: spread throughout
- Caudal: spread throughout
What gene(s) affect the anterior?
What gene(s) affect the posterior?
What gene(s) affect the posterior and anterior?
Torso: transmembrane Tyr kinase receptor
- Maternal gene initially laid down in anterior of egg.
- Transcription factor.
- Activates hunchback
- Represses caudal and nanos
- After fertilization, mRNA is translated and concentration gradient is formed along A/P axis
- Maternal AND zygotic
- A gap gene
- Activated by high bicoid levels in zygote
- Repressed by nanos
- Level of expression activates pair rule by a gradient strongest at anterior.
- Also activates gap genes.
- Spread evenly throughout.
- Inhibition of caudal PROTEIN caused by inhibition of protein synthesis by presence of bicoid.
- Translational regulation
- Represses expression of hunchback.
- Localized in posterior.
Describe the hox gene in flies.
- Highly conserved.
- Located in genome in the same way it's expressed, i.e. first gene at 5' end, last at 3' end.
Why is there a bicoid gradient of expression?
Some genes require a unique concentration to be expressed.
What happens when cytoplasm containing bicoid from a wt is injected into a bicoid mutant?
Wherever the cytoplasm is injected, anterior structures form and neighboring regions develop thoracic segments.
- Transcription factor: mesoderm expression in fly embryo
- Highly conserved: regulates immune system
- Regulated by cactus which inhibits nuclear translocation
- Regulates zygotic genes: twist and snail (ventral), Decapentaplegic (dorsal), short gastrulation (lateral)
- High levels in cytoplasm and nuclei
- Repressed by cactus
Describe Decapentaplegic (Dpp)
- Dorsal region
- Activated in absence of Dorsal
Describe short gastrulation
- Lateral region
- Activated by low amounts of dorsal on lateral cells
If a cell ablation has been performed, how do you tell the difference between cells which fail to differentiate because of lack of cell-cell interactions and those cells which never form because they were in the direct lineage of the ablated cell?
Can have a control of the same set of cells that are not ablated and compare.
What is the advantages of digoxygenin-labeled uracil relative to FITC labeling?
Dig can amplify very low levels. However, once it's used, the sample is done.
When doing an in situ hybridization, what is the appropriate negative control to make sure that the hybridization and RNAse treatment were effective?
Sense probe: gives a measure of non-specific binding due to chemical properties of the probe. If the sense probe detects nothing, then the signal means something.
Why is the RNAse treatment necessary when doing an in situ hybridization?
Minimize noise due to background non-specific binding
Why do we look at mRNA (i.e. in situ hybridization, Northern blot, etc.) when it's the protein that carries the function? Repeated in a later lecture phrased as: Why do people look at both RNA and protein?
There can be a time gap between transcription and translation. mRNA can be sequestered for a time. Other things can happen during this time.
What sort of information can you get from a fate map?
You "know" what to expect normally, so if a change is made, you can compare your results with what you would expect to happen normally.
Why should we care about lineage tracing and fate maps?
Shows relative proportion and arrangement of cells and also their migration patterns.
What are the advantages and disadvantages of a cell transplant experiment?
- Only works in frogs and chicks
- Non-genetic technique
- Simple experiment to see similarities and differences
- But don't know what causes the change
Why is it useless to do a cell transplant to the exact same region?
Presumably, the cells will do the same thing, so you don't know if an effect is due to the transplanted cells or not.
Why do an explant experiment? (Asked twice.) What information will that give you that you don't get through tracers?
- Can eliminate possible interactions with other cells.
- Can physically observe development that you might not normally be able to see
- If you were a frog embryologist, how would you find out of the somites were forming (structures which are inside the embryo and require cell-cell interaction to form properly)?
- Can use a confocal microscope that does "optical sections"
Promised exam question on the first midterm: What characteristics do the genetic models have that the others don't? Also, why are the other models not genetic?
- controlled genetic crosses
- transgenic tools
- system to eliminate/inactivate/introduce a gene
- ability to screen for mutants
Knowing the effect of DmTwist in fruit flies, you see a similar protein present in Xenopus. How do you determine if it has the same function?
RNAi to knock down the expression transiently
If you do RNAi and see no change in phenotype, what should you conclude? How do you test your conclusions?
Test for protein
That was a negative experiment. What would be a non-genetic positive experiment?
Protein injection, or mRNA injection, into cells which don't normally express this protein
Ok, you inject the mRNA and see no change in phenotype. What do you conclude and how do you tests it?
Test for mRNA being present. If there, not the effect you thought it was.
Now you're a mouse embryologist, still studying the effects of Twist. How do you proceed?
Can do knockdown or ectopic expression
If your procedure causes no change in phenotype, what possible conclusions can you draw?
Could be unsuccessful at producing change; twist may not have same function in mouse
Why would you choose a knock-down over a knock-out, and vice versa?
Knockdown is temporary and usually not 100%
Functionally, what information do you get from a laser ablation that you don't get from a toxin ablation, and vice versa?
Laser completely removes other cell, so you lose possible cell-cell interaction
What is the advantage of having, as an investigator, a model that has stereotypic cleavage?
There is a noticeable visual difference between cells which makes fate mapping easier.
How would the cells derived from the M teloblast give rise to muscle? Propose 2 different approaches that you can use to show that the cells in the teloblast give rise to the muscles.
- M cells give rise to mesodermal tissue which give rise to muscle tissue.
- Can do antibody immunostaining
How do the nuclei migrate to the periphery in the fruit fly embryo?
How does the hormonal gradient get established?
Laid down by mRNA which leads to translation of proteins which migrate
In performing a genetic screen on fruit flies for mutations in early developmental genes (things that would be zygotically transcribed), what components should be taken into consideration?
Hm, don't know. Can be done with p-elements, temperature shifts. Males don't have recombination.
Are there mutations in fruit flies which cause the maternal mRNA to be mis-localized?
I'm guessing yes. Perhaps egg-polarity genes
What happens if one of the pair-rule genes are mutated?
Lose segments controlled by these genes - about half
If you are attempting a cytoplasm transfer in fruit fly embryos, what would happen if you injected the wild-type cytoplasm into a separate wild-type embryo?
It depends on where you inject it, but likely nothing if it's in the same spot, but it could have an adverse affect since overexpression can be problematic too.
If a bicoid mutant fails to make bicoid, what would be the outcome?
Loss of anterior/head portion
Explain why, in order to activate DmTwist, high concentrations of Dorsal are required. Why is this system better than, say, using protein degradation to establish a gradient and meet threshold requirements?
- Has multiple binding sites
If Dpp is removed, what will be the outcome?
No extraembryonic or dorsal epidermis tissue
What would happen (would it even be possible) to reverse the normal gradients, i.e. dorsal cells release Sog and lateral cells release Dpp?
Not possible according to my notes for some reason.
What are the necessary components of a useful p-element?
Promoter, gene of interest, recognition sequence
What can p-elements be used for?
Inserting gene or knocking out genes
What is the most appropriate inducible promoter for p-elements in flies?
Propose a p-element experiment with an inducible promoter to show that Dorsal will repress the production of Dpp.
Can express Dorsal where it's normally not, but where Dpp normally is.
Say that you successfully incorporate the above p-element into the genome of the fly, and you have done immunostaining to show that the protein is being expressed everywhere, but Dpp still gets formed. Explain this outcome.
Propose a molecular approach you can use to do the same.
How does the hunchback mRNA and protein play a role in the threshold component of fruit fly development?
Pair-rule genes are regulated by the expression of hunchback gradient
In a fruit fly embryo, if you saw that the early stripes failed to develop, what would you conclude had happened? What are the possible causes of this?
Lack of striping can be due to any earlier steps in development, so bicoid, gap genes such as hunchback, or pair-rule genes.
What are some of the differences between leeches and flies for cell cleavage?
Leeches have multicellular embryos early on unlike flies which have a multi-nucleated embryow. Cell cleavage in leeches is not symmetric, i.e. spiral cleavage.
How can a western blot be quantitative?
Can use fluorescence and quantitatively measure that fluorescence.
What information do you get with a transplant vs an explant?
Transplans shows ectopic expression; sufficiency.
What do you get from toxin ablation that you don't get from laser ablation?
Can still see possible cell-cell (or cell-machinery) interaction
If you do a knockdown and see no phenotype, what are the possibilities, and how do you test them?
Could have failed. Could be redundancy. Can check for protein.
What makes a model a genetic model? (You're going to get that for sure.)
- Transgenic tools
- Ability to eliminate/inactivate/introduce mutation in a gene
- Ability to do controlled crosses
- Ability to screen for mutants