Name: _______________________________ 2007 7.013 Problem Set 5 Due before 5 PM on WEDNESDAY, April 18, 2007. Turn answers in to the box outside of 68-120. PLEASE WRITE YOUR ANSWERS ON THIS PRINTOUT. Question 1. The limb (arm or leg) forms when a group of cells called “lateral plate mesoderm” (LPM) induces overlying ectoderm (EC) to form an outgrowth called the “limb bud”. The limb bud forms an outgrowth because the number of cells it contains increases relative to surrounding ectoderm. 1a. One possibility is that cells in the bud undergo increased cell division relative to surrounding ectoderm. What technique could you use to ask whether this possibility is correct? (5 words or fewer) 1b. Give another possible mechanism that would increase cell number. (5 words or fewer) 1c. Interestingly, part of the brain (the “hindbrain”) can substitute for the LPM and induce limb bud outgrowth. Discuss the molecular biology that is likely to explain this result (15 words or fewer). 1d. Young lateral ectoderm can give rise to many fates, including the limbs, brain and spinal cord, the epidermis of the skin, pigment cells. However, in older embryos, the lateral ectoderm can form only a limb bud. What is the term for the possible fates that a cell can form? Forelimb Bud(Tbx5)Hindlimb Bud(Tbx4)LPM EC LPM ECName: _______________________________ The front and back legs of the chicken are very different, where the front limb is covered with feathers, and the hindlimb is covered with scales. In order to figure out why these limbs are different from one another, the following transplant experiment was performed. LPM from the hindlimb area was placed under future forelimb ectoderm, and LPM from the forelimb region was placed under future hindlimb ectoderm. The resulting chick embryos were assayed after the limbs had differentiated, and the following results were obtained. future forelimb LPM + future forelimb ectoderm feathers future hindlimb LPM + future forelimb ectoderm scales future hindlimb LPM + future hindlimb ectoderm scales future forelimb LPM + future hindlimb ectoderm feathers 1e. What do these data tell you regarding the mechanism by which fore- and hindlimbs make feathers or scales? (15 words or fewer) Chickens have white feathers, quail have black feathers. You perform a transplant experiment like the one above, as detailed below, with the following results: Chick forelimb LPM + chick ecto white feathers Quail forelimb LPM + chick ecto white feathers Chick forelimb LPM + quail ecto black feathers Quail forelimb LPM + quail ecto black feathers 1f. What do these data tell you regarding the mechanism by which chick and quail make different color feathers? (15 words or fewer)Name: _______________________________ The Tbx4 transcription factor is expressed exclusively in the developing hindlimb, and is necessary for development of hindlimb characteristics, including whether it has scales or feathers. A mutant has been isolated where the hindlimb now looks like a forelimb. Linkage analysis indicates that this mutant lies in the Tbx4 gene. However, in order to confirm mutant gene identity, you sequence the Tbx4 genomic DNA from this mutant, and find that there are no changes in any exon. On the other hand, you find two nucleotides in the mutant DNA, which differ from wild type. The first is at the exon1/intron1 splice acceptor sequence. The second is 25 bp 5’ to the transcriptional start site, where the wild type sequence 5’ TATAAAAT is instead 5’TATAGGGC. 1g. In the splice acceptor mutant, what changes would you observe in the RNA made from this gene, relative to wild type? (15 words or fewer) 1h. Could this change account for the Tbx4 mutant phenotype? Explain in 15 words or fewer. 1i. If the mutant that maps to the non-transcribed part of the gene (TATAAAAT), what changes would you observe in the RNA made from this gene, relative to wild type? (15 words or fewer). 1j. Could this change account for the mutant phenotype? Explain in 15 words or fewer. 1k. Tbx5 is required for forelimb development, and normally expressed only in the forelimb. In the Tbx4 mutant, you find that Tbx5 is now expressed in both sets of limbs. Explain (use a diagram if you like).Name: _______________________________ Question 2. 2a. Tube formation is often accompanied by lengthening or extension of an initially short tube. What two processes lengthen a cell sheet? Explain what each entails (15 words or fewer). (i) (ii) The neural tube forms the brain and spinal cord. Wnt signaling is required for lengthening the tube. The future neural tube lies next to the notochord and the somites. 2b. Give two ways you could determine where the Wnt signal comes from? Your answers should be independent, and include… (i) use of an explant assay (ii) use of gene expression assays Formation of the neural tube involves a sharp ventral bend as diagrammed. This bend is associated with formation of wedge-shaped cells to form the “floorplate”. 2c. One gene which is expressed in the floorplate is “sonic hedgehog” (shh). How could you determine whether shh is necessary for floorplate formation? (15 words or fewer) 2d. What structural cytoskeletal protein would you expect to change in the floorplate cells as they become wedge-shaped? Somite SomiteNotochordSomite SomiteSomite SomiteFloor PlateNeural Crest MigrationName: _______________________________ 2e. How would this protein change? 2f. The compound “blebbistatin” inhibits myosin function. If you applied blebbistatin to the developing floorplate, what effect would you expect to observe? Explain in 15 words or fewer. 2g. As shown in the above diagram, the floorplate lies above a structure called the “notochord”. shh is also expressed in the notochord, and is responsible for inducing expression of shh in the floorplate. How does shh expression in the notochord activate shh expression in the floorplate? (15 words or fewer) As diagrammed above, from the dorsal neural tube, an important group of cells called the “neural crest” begins to migrate to many parts of the embryo. When they reach their final destinations, they differentiate into pigment cells, the bones and muscles of the face, the
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