This next assignment was Problem Set 1, which was given to us during the third week of classes. Here, we studied maternal effect genes, the A-P and D-V asymmetry. We also looked as some cool effects and consequences when concentration gradients are skewed incorrectly, such as mRNAs gurken, oskar and bicoid which are all normally asymmetrically distributed in the cytoplasm - very bad for drosophila, poor things!
Patterning in the early fly embryo
1. Drosophila embryos produced by torso mutant mothers lack their terminal parts (acron and telson), regardless of the genotype of their fathers. Homozygous torso-/torso- embryos from heterozygous mothers develop normal acrons and telsons. What conclusions can be made about:
a) The nature of the torso gene? it is a maternal effect gene since all embryos from mutant mothers are affected
b) The function of the torso gene? it is a response to or is necessary for development of terminal parts (acrons and telsons)
By now you know the molecular nature of the Torso protein. Imagine that you did not have that information, and you thought it could be either a soluble, diffusible protein (like Spz or Bcd), or a membrane-associated protein. What experiment/manipulation could help you distinguish between these two possibilities?
c) If it were a diffusible protein, what would you expect to observe?
If it was diffusible protein, we can try transferring cytoplasm to a different cell to see what effect.
- Do a rescue experiment to see if you can transfer cytoplasm to give the phenotype
- Do cytoplasm fractionation.
d) If it were a membrane-associated protein, what would you expect to observe?
If it was a membrane protein, transfer the membrane and see what effects it has. If the protein does not function without the membrane, we know its a membrane protein, and if it does we know it is independent of the membrane.
2. In loppins (small, diploid animals increasingly popular in several genetics courses), five different LOF mutations in the nan gene have been isolated. Homozygosity for any of the LOF nan alleles results in a complete lack of antennae.
a) What can you conclude regarding the function of the nan gene product?
It is required for the formation of the antennae
b) Based on the current loppins literature, you suspect the Nan protein to be part of a larger, multi-protein complex that also contains the protein Js. Your colleague, on the other hand, is convinced that Nan acts alone and downstream of the Js-containing multi-protein complex, in a signalling pathway.
i) If you are correct, what should you observe in a nan mutant with respect to the Js protein? (Think about the distribution/localization of the protein and well as its concentration and function).
Both phenotype of nan mutant and Js mutant would have same phenotype. Use EMSA to determine whether or not they form a complex.
ii) If your colleague is correct, what should you observe in a nan mutant with respect to the Js protein?
The Js protein should stay the same, if Nan is independent AND downstream of Js
iii) Assuming that your ‘suspicion’ (i.e. hypothesis) is correct, predict the phenotype of a js- (complete LOF) mutant (think about as many aspects of its phenotype as you can, such as general morphological aspect, gene expression patterns, etc).
Complete lack of antennae, which would be the same as nan- mutant, since I believe that Js is dependent of Nan
iv) Would your prediction be different if it turned out that your colleague’s hypothesis, and not yours, is the correct one?
If not, what experiment could you do to determine which hypothesis is correct?
Predict the outcomes of your experiment if your hypothesis was correct and if your colleague’s was correct.
You can do a loss of function of Nan with wildtype Js, and compare this phenotype to a loss of function Nan and Js to determine whether Js alone alters the phenotype. If they show the same phenotype, it is likely that Js is dependent of Nan, however it is possible for the phenotype to be the same, assuming that the Js doesn’t change the phenotype by itself.
Patterning in a vertebrate limb bud-refer to the attached figures and to the Bastida and Ros (2008) review paper for help, information and inspiration.
3. SHH s essential for a number of processes (pretty much everything…). What do we need to be able to do if we want to study its role in limb development, specifically?
Make lots of mutants with LOF of SHH receptors, and identify the phenotypes individually. Stain the SHH and identify the localization. Use RNAi to try and disrupt the mRNA expression at a controlled stage.
4.a) What would be the phenotype of an individual with a complete LOF mutation in Shh in the cells that give rise to the limbs? Propose a reasonable hypothesis.
The individual would not have any limbs since SHH is responsible for patterning and development of most limbs.
b) In fact, researchers have carried out this experiment, and they observed that the individuals with this mutation had abnormal autopods (including absent or severely malformed d2-d5), yet their first digits were developed normally. What can we conclude from this information?
Digit 1 is independent of SHH, but is necessary for d2-d5
5. If we injected lots of SHH in the anteriormost region of a chicken's limb bud, what phenotype should we obtain? (Explain your reasoning)
Dependent on which stage we inject the SHH. SHH are very time dependent, location dependent, and concentration dependent.
6. Predict what phenotypes we would see if we stopped the limb bud expression of Shh precisely, at the following time points:
a) Very early in the development of the limb bud
Loss of posterior digit
b) About halfway through the development of the limb bud
Malformed digits that are associated with Shh, very time dependent.
c) Late in the development of the limb bud –
Possibly no effect, dependent of the time
Amanda
email: manda147@interchange.ubc.ca | Student Number: 92681071
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