Problem Set 2 Induction in C elegans embryos Zoology 470 Spring 2015 20 Points Total Problem Set Guidelines 1 Due date This problem set is due by the end of class on Wednesday March 25 2015 2 Sources You may use any sources at your disposal to answer the following questions Legitimate sources include classmates knowledgeable friends and colleagues written documents and any other scientific resources you find useful If you work with other classmates on this problem set we ask that you list the other students with whom you worked to answer these questions Although you may discuss these questions as part of a group you are expected to answer the questions as an individual If you believe that published references will help you answer these questions you may cite those references However citation of additional references is not required nor is it expected 3 Answering the questions This problem set is designed to be answered concisely Brief but complete answers should be written in the space provided It is acceptable to type your answers but you must provide a hard copy of the document you generate Where you are asked for explanations you must provide them to receive full credit If you find it helpful feel free to include diagrams in your answers You need only turn in your answers on pages 2 3 Necessary information All of the information and techniques needed to answer the questions on p 2 3 have been presented in class or are to be found in Gilbert s Developmental Biology This problem set requires you to learn about a new signaling pathway the Notch Delta pathway which is described in Chapter 3 of Gilbert p 98 You may also find it helpful to review your reading on C elegans early development from Ch 5 Problem Statement Several labs beginning in the 1980s began investigating inductive interactions that lead to differentiation of the anterior most cell from the four cell embryo AB a and its posterior sister cell AB p These two cells give rise to cells of the pharynx a mesodermal structure Normally AB a generates cells that form the anterior pharynx while AB p normally gives rise to cells that make the posterior pharynx These two types of cells express different proteins and differentiate quite differently There are several papers that you are free to consult However it is not necessary to read these papers to answer the questions on pages 2 3 Papers that may be relevant include the following which can be downloaded from Learn UW Priess J and Thomson N 1987 Cellular interactions in early C elegans embryos Cell 48 241 50 Mello C C Draper B W and Priess J R 1994 The maternal genes apx 1 and glp 1 and establishment of dorsal ventral polarity in the early C elegans embryo Cell 77 95 106 Mango S E Thorpe C J Martin P R Chamberlain S H and Bowerman B 1994 Two maternal genes apx 1 and pie 1 are required to distinguish the fates of equivalent blastomeres in the early Caenorhabditis elegans embryo Development 120 2305 15 Evans T C Crittenden S L Kodoyianni V and Kimble J 1994 Translational control of maternal glp 1 mRNA establishes an asymmetry in the C elegans embryo Cell 77 183 94 Zoo 470 2015 Problem Set 2 Page 2 Name Student Number If you worked in a group other collaborators 1 Testing how AB a and AB p differentiate Recall that there are four cells in the C elegans embryo AB a AB p EMS and P2 whose spatial relationships are shown correctly at the right anterior is to the left in the diagram a Jim Priess Craig Mello and Bruce Bowerman developed ways to manipulate blastomeres in the early embryo They found they could move cells around by pushing them with a blunt needle so that AB a could be moved to lie in the position normally occupied by AB p They also developed ways to remove cells from the embryo by sucking them out of the eggshell with a pipette a 4 points Jim believed that P2 normally sends an inductive signal to the AB daughter it is touching that leads that AB daughter to form posterior pharynx Using the simple blastomere manipulations Jim developed describe one experiment you could perform to show P2 contact is sufficient to induce either AB daughter cell to form posterior pharynx b 4 points Craig Mello and Jim Priess created the embryo below on the right b A normal embryo is shown for comparison a If Jim s hypothesis is correct will this embryo make more or less anterior pharynx than normal Circle the correct answer Embryo b will make more the same less anterior pharynx than normal Clearly state your reasoning 2 apx 1 glp 1 and pharynx differentiation a 2 points Craig and Bruce working independently showed that a gene called apx 1 for anterior pharynx in excess is required for posterior pharynx differentiation apx 1 is a maternal effect gene If a mother hermaphrodite worm has the genotype apx 1 wild type copy of the gene i e it is a heterozygote what percentage of its offspring will have defective pharynges pharynges is the plural of pharynx Percentage of offspring from apx 1 with defective pharynges Zoo 470 2015 Problem Set 2 Page 3 2 cont b 4 points Please refer to p 96 of Gilbert 10e for information on the Notch Delta pathway Craig showed using a special mutant in the glp 1 gene that glp 1 is needed for correct differentiation of AB p glp 1 encodes a Notch protein Craig went on to show that apx 1 encodes a Delta family protein You are now working in Bruce Bowerman s lab where techniques were developed for isolating blastomeres and recombining them as we discussed for Wnt Frizzled signaling in class You are eager to try blastomere recombinations using cells lacking either apx 1 or glp 1 function You assess anterior pharynx formation using an antibody that recognizes anterior pharynx Based on your study of Notch and Delta what would happen if an AB p cell lacking APX 1 protein were combined with a P2 cell lacking GLP 1 protein Clearly explain your answer 3 Translational control of glp 1 a 1 point Tom Evans who was working in Judith Kimble s lab here at UW at the time showed that glp 1 mRNA is found everywhere in the four cell embryo but that the protein is only found in two cells in the four cell embryo What technique did he use to assess mRNA localization Technique b 3 points Tom hypothesized that the glp 1 mRNA is subject to translational control To see which part of the mRNA was involved he used molecular biology techniques to make a piece of DNA that encoded a reporter protein he could detect galactosidase fused to DNA encoding parts of the glp 1 mRNA He then assessed whether