STUDY QUESTIONS FOR THE DISCUSSION SESSION OF Monday, January 20th 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) ARTICLE 2 (Mutations affecting segment number and polarity in Dropsophila) What was objective of study? Investigation of mutations that influence pattern of segmentation in Drosophila larva that depend on genome of the zygote - Objective: find all genes that are involved in polarity of segment, number of segment, organization of segment How did the researchers proceed? - 1) Started with adult male fruit flies and perform mutagenesis on them (use male because dont mess with female because she makes eggs and if effect how she makes eggs properly then no embryo) - 2) mate with ~WT female progeny; some heterozygous mutants self-cross progeny (some of these individuals will be homozygous for mutation and these are the ones that researched are interested in) - 3)in this homozygous progeny look for phenotype regarding segmentation at stage of young larvae/embryo What experimental approaches did they use? - Complementation testing(check): to see which allele associated with which gene if 2 mutant complementary in same genes , cross two mutants with similar phenotypes and observe if progeny has similar phenotypes - Mutant need to be recessive to wild type (cross them together should get 100 percent mutant in offspring) 100 percent heterozygous if mutant are recessive and should get WT phenotype - Take all mutants with same phenotype and cross them in pairs to determine how many dif genes they hit and how many dif alleles they had identified 10 dif mutants with same phenotype cross them Progeny give WT phenotype = 10 unique genes are involved Progeny with mutant phenotype = 10 alleles of the same gene (i.e. 10 dif mutants with same phenotoy and if after cross and they all completemneach other they alwas give you mutant then theygive you 10 allele of same gene but if give WT phenotype then you know have 10 dif genes What were the results? Mutations at 15 loci were found categorized in 3 phenotype: a) Segment polarity mutants: - normal number of segments - Defined portion of pattern in each segment is deleted = results in mirror-image duplication in each segment - 6 loci identified Fused,Wingless, cubitus Interruptus, Gooseberry, Hedgehog, patch b) Pair-rule mutants - pattern deletion in alternating segments - 6 loci identified (each with specific pattern) Even-skipped, odd-skipped, paired, runt, engrailed, barrel - Mutant phenotypes show embryo structured in repeating units c) Gap mutants - deletion of large sub region with 8 adjacent segments - 3 loci (kruppel, knirps, hunchback) What was novel about each paper? - First time rigourous genetics screen had been used to discrover genes in development - Found genes involved in Intermediate develop i.e. number of segments and organization of segments- first time these genes were discovered --- prior to this genes for early development and later development had been found

Found relatively small number of genes prior to this was thought there would be thohusands of genes and thought task would be impossible Mutant phenotypes only seen in homozygous embryo = 15 loci are active following fertilization 15 loci influence segmentation in Drosophila genome how did the authors determine allellism? - Complementation testing Why is it important to know how many different mutant alleles of a gene were identified in the screen? TABLE 1: linkage analysis performed to find map position; found number of alleles in each gene - important to find number of alleles to see if have identified all of the genes - important to show that each gene has been hit multiple times to confirm that there are no other genes to hit = more genes you ave that have been hit multiple times would increase validity - more confidence that our screen is close to saturation = have caught most of the genes needed (using statistics) That mutations are embryonic or larval lethal (homozygous lethal). How do you think the authors managed to keep such mutants in their stock? - Keep stocks of hetereozygous for mutants and perform crosses to obtain homozygous mutants

ARTICLE 3: (Identification of genes with unique and essential functions in the development of the zebrafish, Danio rerio 1163 mutants (in 372 genes) isolated using genetic screening in embryos or early larvae of zebrafish - Mutations must have visible phenotype: mutations found in body axes, jaw and gills, pigment cells, organs, CNS, mesoderm - genes found involved in: morphogenesis, pattern formation, differentiation and organ development Complementation crosses between mutant pairs with similar phenotypes performed - Majority of mutants show more than one phenotype (multiple alleles per gene) Overally allele frequency average = 2.4 - Average allele frequency in different phenotypes = 1 7.5 Majority of 372 genes correspond to only 1 allelle 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? Did they combine multiple approaches? ARTICLE 2: staining ARTICLE 3: mutagenesis 3) Refer to paper #2 (the longer Drosophila paper) Back in 1980, what set the authors approach apart from other developmental biology/developmental genetics studies? (Whats novel about their study?) Conducted experiements to identify genes involved in development of Dropsophila embryo Not a lot was known about genetic mechanisms of how multi-cellular organisms develop during embyrogenesis Now consider paper #3 (the zebrafish paper): Whats novel, and whats extremely valuable about paper #3? Exceptionally large number of genes identified in zebrafish using genetic saturation screen By using zebrafish embryo, phenotype of internal organ development could be observed easier Dipolids were screened Prior to this paper (#3), Dr Nsslein-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 projects chances of success? What would do you think would be the main challenges? Development of zebrafish is significantly more complicated than dropsophilla = exhausting process and life cycle is longer Barely find genes in drosophilla and now tackle vertebrate of fish looking for all developmental genes not just segmentation = crazy concept Thought fish had same number of genes as humans 4) What general conclusion can be made from the results presented in papers #2 and #3? Article 2: mutants that affect axis determination and segmentation in Dropsophilla embryo results in a small number of pattern-forming pathways Article 3: 5) In paper #3, the researchers decided to do a DIPLOID screen, as opposed to using haploids (as depicted in Griffiths MGAs 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 MGAs figure 12-24 to Figure 3 in paper #3). Advantage: process of identifying mutants in different phenotype classes is more consistent and reliable - Haploid: associated with developmemental problems haploid phenotype difficult to disticguish from mutant phenotype (due to mutation from gene vs from being haploid) Disadvantage: takes more work to cross - Haploid: no crossing would be needed Advantages: - Haploid (1n screen) : observe mutants in progeny earlier ecuase one less generation but cant isolate mutants [n

a haploid screen (a), female F1 fish (derived from the cross between a wild-type female and an ethylnitrosourea (ENU)-mutagenized male) are squeezed gently to release their eggs, which are then fertilized with ultraviolet (UV)-treated sperm to generate haploid embryos. UV treatment destroys the parental DNA, without affecting its ability to activate the egg. A haploid clutch derived from a heterozygous female will contain 50% mutant and 50% wild-type embryos. ]
Diploid (2n screen): see one generation later (due to needing to make homozygous) but it will be isolated; Can keep heterozygous stocks with parents but cant with haploid Disadvantages - Haploid: cant differentiate between dominant or recessive mutation; manipulating and working with artificial background ::cant recplicate with diploid due to artificial manipulation -

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)? To categorize similarities easier and present strong support and evidence 6) Study Paper #3s Table 4. What does it show? Be prepared to discuss the information shown in the table in a lot of detail!

Majority of 372 genes (222) correspond to only 1 allelle 11 genes: 11 34 allelles governing Fewer genes have more than 10 alleles Number of allelles in genes decrease as number of alelles increase Table shows number of alleles found in gene screening (i.e. 12 genes found with 5 allelles) Mutants = multiple allele with genes shows each mutant has different allele o Assume: Close to saturation can be calculated by seeing number of genes hit

7) a) Genetic screens are probably the oldest genome-wide experiments around. What is another genome-wide type of experiment that you know of? b) Compare the type of information provided by a genetic screen vs. your other genome-wide experiment. c) Think about the obvious follow-up experiment(s) for a genetic screen and for your other genome-wide experiment. Are they the same or different? How do they compare? d) Would you say that your other genome-wide experiment is a reverse genetics or a forward genetics one? 8) What kinds of mutations did the authors of article #2 identify? Mutants that effect segmentation and axis determination in Drosophilla 9) 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). What are two potential explanations for the multiple mutant phenotypes of dak mutants? o Pleiotropy mutation in one genes influence many phenotypic traits o 10) Imagine that its 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 youd 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!] 11) 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? Suggestions: -SAVE A COPY OF YOUR ANSWERS -SAVE A COPY OF WHATEVER NOTES (RELATIVE TO THE ASSIGNMENT) YOU TOOK IN CLASS -SAVE A COPY OF ANY COMMENTS/QUESTIONS YOU POSTED ONLINE Why? Because they make a great piece for your individual portfolio (one of your five required assignments to include)! You may also find that material in this assignment lends itself to being a piece of evidence to show your learning in your Learning Journal!

Results of an experiment = shell ask for conclusion

Learning journal: describe what learn, add piece of assignment [get piece of note that is relevant], use resources and activites