Assignment #4

Assigned articles:

1)  Schubiger, G. (1980) The development of animal segments.

(Note that this short article is an introduction to/presentation of the second article).

2)  Nüsslein-Volhard, C. and Wieschaus, E. (1980) Mutations affecting segment number and polarity in Drosophila.

3)  Haffter, P., et al. (1996). The identification of genes with unique and essential functions in the development of the zebrafish, Danio rerio.

Questions

1)  Briefly summarize articles #2 and #3 IN YOUR OWN WORDS (point form is fine… maximum 250 words in total for the two papers)

In articles 2 and 3, using Drosophila and Zebrafish, respectively, they explained experiments regarding mutations that affect development. The drosophila paper illustrated how segment polarity mutants, pair rule mutants and gap mutants cause phenotypic alterations. These 3 classes are responsible for embryo defects in their subregions on repeat length. With the zebrafish in paper 3, experiments were preformed to find the phenotypic alterations in embryos using mutagenic treatment to mutate specific genes in attempt to characterize genes responsible for segmentation and growth. This allowed the researchers to find mutants in 372+ genes, with a wide range of mutated phenotypes.

2)  Think about the kinds of experimental approaches that were discussed in class. What kind of approaches did the authors take in the experiments reported in papers #2 and #3?

Primarily loss of function experiments for papers 2 and 3:

     Paper 2: This was shown in deletion of segments which result in a mirror-image duplication -> fused, wingless, cubitus, inturruptus, gooseberry, hedgehog and patch mutants came from a loss of function experiment that deleted different segments of DNA.

     Paper 3: This was evident because they only mutated parts of the genome, resulting in loss of function of certain genes to find if some genes are necessary for function or development in embryogenesis.

No gain of function experiments were done for either papers.

3)  Refer to paper #2:

Back in 1980, what set the authors’ approach apart from other developmental biology/developmental genetics studies? (What’s novel about it?)

They were able to isolate and represent the majority of the loci affecting segmentation from the entire drosophila genome, thereby identifying all the genetic components involved in embryonic pattern formation

Now consider paper #3:

What’s novel, and what’s extremely valuable about paper #3?

They were able to conclude that the most severe limitation of detection of genes with important functions using mutational approaches (which is what their experiments were based on) was redundancy. They showed that there was complete or partial overlap in functions of two or more genes involved in the same process.

Prior to this paper (#3), Dr Nüsslein-Volhard publicly presented her intention to do with the zebrafish what she and Wieschaus had done with Drosophila in terms of identifying all the genes required for early development. People did not laugh in her face because she was extremely well-respected and well-known for achieving what she set out to do, but many a researcher quietly questioned her sanity, as the project seemed to be an extremely challenging one.

Why do you think other scientists were skeptical about the project’s chances of success?

Because the number of genes in zebrafish, compared to drosophila, is much larger. The sheer number of screenings and mutants that she and her researchers had to conduct was enormous in such an ambitious study. Of 3857 mutagenized genomes, all were extensively screened and only 1163 were kept and characterized.

4)  What general conclusion can be made from the results presented in papers #2 and #3?

A general conclusion can be that embryogenesis is extremely complex and characterized by many genes that each have both an individual function and functions as part of a larger group of up or downstream pathways.

5)  In paper #2, the authors’ hypothesis is not clearly stated. Nonetheless, they did have one. What do you think it was? (Hint: you may find valuable information in this sense in paper #3!).

I think their hypothesis was that loss of function phenotypes can show genes that are essential for development and pattern formation such as segmentation.

6)  In paper #3, the researchers decided to do a DIPLOID screen, as opposed to using haploids (as depicted in MGA’s Figure 12-24). What are the advantages and disadvantages of doing a diploid screen, as opposed to a haploid one?

(It may help to compare MGA’s figure 12-24 to Figure 3 in paper #3).

Doing a diploid screen was done in paper 3 because the identification and recovery of mutants of many phenotypic classes can be performed with more consistency and reliability. However, it’s much more laborious and time consuming.

7)  In both paper #2 and paper #3, the authors performed a set of complementation tests. Why did they test mutants with similar phenotypes against each other (as opposed to testing every single mutant against all of the mutants)?

They did this to identify the mutant genes, to see whether mutants that displayed the phenotype showed the same genotype because it’s more likely that there would be a correlation in such case. Furthermore, it would be much more time consuming to screen all mutants against one another.

8)  Study Paper #3’s Table 4. What does it show? Be prepared to discuss the information shown in the table in a lot of detail!

This table shows the number of alleles, number of genes and number of mutants that were found. They demonstrate that the allele frequencies that are observed were not random because there are significant numbers of mutants that correspond to each allele and gene.

9)  a) Genetic screens are probably the oldest genome-wide experiments around. What is another genome-wide type of experiment that you know of?

Reverse genetic screens are for discovering the function of a gene by analysing the phenotypic effects of specific gene sequences. This is like the opposite of genetic screening. An example is creating KO mice of any sort to analyse phenotype.

b) Compare the type of information provided by a genetic screen vs. your other genome-wide experiment.

Genetic screens show the genome of individuals with a particular phenotype, therefore you can find SNPs or other mutations in the gene. Reverse genetics show the phenotype of a gene that you alter so you can find the effects of mutating or silencing a particular gene.

c) Compare the obvious follow-up experiment(s) for a genetic screen and for your other genome-wide experiment.

Follow up to genetic screens would be to characterize the SNPs, and screen further subjects. For reverse genetics, alter the gene in another way, or transfect another individual (mouse) with the gene and see if the same effect occurs

d) Would you say that your other genome-wide experiment a “reverse genetics” or a “forward genetics” one?

It’s a forward genetics one because it identifies the genes responsible for a phenotype, like finding the underlying cause of a big picture, whereas reverse genetics starts with genes and finds phenotypes.

10)   Think about the types of mutations discussed in class, and also about the kinds of mutations described in Figure 12-38 of your background reading for this week. With respect to those examples, what kinds of mutations did the authors of article #2 identify?

They found segmentation mutants; segment polarity mutants, pair rule mutants and gap mutants cause phenotypic alterations in segmentation of the abdomen and thorax.

11)   In paper #3, several mutants have multiple phenotypic defects (see for example Table 5). For example, defects in the otic vesicles are reported to be often accompanied by other mutant phenotypes, such as lack of the pelvic fin. What does this suggest?  (Think about at least TWO possible explanations for this phenomenon).

It suggests that otic vesicles and the pelvic fin (for example) are dependent of each other. This could be due to the affected gene being an upstream of the other, therefore both are affected when the upstream gene is not functioning, or they are both part of a multi-system complex that cannot function independently.

12)   Imagine that it’s 1980, and you have just come into possession of one of the mutants described in article #2 (choose your favourite one). You are interested in the control of Drosophila development and are planning to apply for a new research grant. List 3 important questions relating to your mutant of choice that you’d like to tackle over the next couple of years.

[You are encouraged to post your answer to this question on the bulletin board and discuss it with your classmates!]

For gap mutants: At what stage in development is the A-P axis determined? Is kruppel dominant or recessive? Is kruppel a maternal-effect gene? What happens if there is overexpression of kruppel?

13)   Based on what you have learned from the assigned articles, and on your own experience and knowledge, what is the relevance of discovering the relationships between specific mutations in specific genes and the specific phenotypes associated with them?

This helps us to understand embryogenesis and what genes are important for this. It allows us to associate specific genes with certain phenotypes, and determine the necessary concentration, location, and timing for the transcription/translation of cretain genes in order for healthy development.

Amanda

email: manda147@interchange.ubc.ca | Student Number: 92681071