Answering long questions
Biology Name / pledge: 1. In Drosophila, mRNA of the maternal effect gene gurken is localized in the dorsal region of eggs, while a second protein-coding gene, pipe, is normally expressed only on the ventral surface of eggs. Eggs with wild-type expression of gurken and pipe eventually develop normal dorsal and ventral embryonic structures in the appropriate places. However, female flies homozygous for a gurken null mutation lay eggs that lack functional gurken mRNA, and the region of pipe expression expands to include the surface of the entire egg. Such embryos are unable to develop dorsal structures, and ventral structures develop on both sides of the early embryo. (See image at right, which shows cross-sections of eggs and early embryos of both wild-type and mutant flies.) A. What does the information above suggest about the normal regulatory relationship between gurken and pipe? Briefly explain (2 points). B. What does the information above suggest about the normal roles of gurken and pipe in the development of dorsal and ventral structures? Briefly explain (2 points). C. Imagine that you are able to isolate gurken mRNA. How could you use this mRNA to experimentally test the regulatory hypothesis you proposed in (A)? Briefly explain your proposed experimental test (2 points). D. Three other protein-coding genes downstream of gurken are also involved in controlling pipe expression: capicua, mirror, and torpedo. Here is information about the phenotypic effects of mutations in these genes: ● Mutants lacking capicua, no pipe expression ● Mutants lacking mirror, expanded pipe expression ● Mutants lacking torpedo, expanded pipe expression ● Double mutants lacking both capicua and mirror, expanded pipe expression ● Double mutants lacking both capicua and torpedo, no pipe expression Using the arrow and T-bar system (you may copy and paste the → and⟞ symbols as needed), complete the developmental regulatory pathway that controls the expression of pipe in the space below (6 points). E. The protein products of capicua and mirror are both transcription factors; now propose a hypothesis that is consistent with the developmental pathway you proposed in (D) that would explain how capicua and mirror could modify pipe expression by acting as transcription factors, and briefly explain your hypothesis (3 points). 2. You have identified a DNA cis-regulatory element (CRE, or enhancer) that is responsible for the expression of the Drosophila gene even-skipped in stripes 3 and 7, as it can switch on expression of a reporter gene (blue) in those stripe regions (panel A). You next decide to test this CRE in various mutant embryos, and observe reporter gene expression in each of those embryos. Page 2 A. You begin with a tailless mutant embryo (Panel D). Based on the observed pattern of expression, what do you conclude is the normal role of tailless in regulating this CRE, and why do you conclude that (2 points)? B. You next observe reporter gene expression in a knirps mutant (Panel B). Based on the observed pattern of expression, what is the normal role of knirps in regulating this CRE, and why do you conclude that (2 points)? C. Finally, you observe expression in a mutant lacking both knirps and torpedo (Panel E). Based on the observed pattern of expression, what is the normal role of torpedo in regulating the CRE, and why do you conclude that (2 points)? 3. During development in vertebrates, mesodermal cells near the neural tube become organized into blocks of tissue called somites. Initially the cells that comprise the somites are undifferentiated and appear identical to each other. Later in development, however, the cells begin to differentiate and become either connective tissue, muscle, or bone (see diagram of somite at right). Describe an experiment you could use to decide whether cell fate in the somites is determined or undetermined at the early stage of somite development when all cells appear identical. Briefly explain your expected outcomes and how you would interpret them (4 points). Exam continues on next page 4. During the early stages of mouse lung formation, cells in the lung epithelium (a layer of endodermal cells) divide and form primary (1o) bronchial tubules which then branch and form additional secondary (2o) Page 3 and tertiary (3o) tubules. The epithelial cells overlie, but are not in direct contact with, a different cell type, the lung mesenchyme. If the lung mesenchyme is either removed or replaced with kidney mesenchyme, the cells of the epithelium do not divide, and bronchial tubules do not form. A. Why do the results described above indicate induction via a diffusible signal? Briefly explain, and include in your explanation a brief definition of induction (2 points). B. Further study shows that lung mesenchyme cells secrete two diffusible signal molecules that kidney mesenchyme cells do not: Fibroblast Growth Factor (FGF) and Bone Morphogenic Protein (BMP). Using what you have learned about how experiments with FGF have clarified its role in the development of vertebrate limbs, describe how you could experimentally and conclusively determine whether just one or both of these signal molecules are required to induce bronchial tube formation in the lung epithelium. Briefly explain your proposed experimental procedure, and briefly explain how it would allow you to make this determination (3 points). C. Suppose that after performing the experiment you designed in (B), you acquire conclusive evidence that FGF, and not BMP, is the diffusible signal molecule involved in bronchial tubule formation. FGF performs its role as a signal molecule by binding with a receptor kinase. Where would you expect to find FGF receptors in the lung cells described above, and what is likely to be the end result of FGF binding with the receptor? Be sure to include in your answer the cell type where you would expect to find the FGF receptor, its expected subcellular location within the cell, and how FGF binding with the receptor might lead to altered lung development (3 points). D. Altered FGF signaling has also been implicated in certain types of lung cancers; would you expect FGF to be underexpressed or overexpressed in cells that are part of lung cancer tumors? Briefly explain (2 points). E. Min et al. (1998, Genes and Development 12:3156-61) generated mutant FGF-deficient mice in which the gene for FGF had been deleted. What predictions can you make about both lung development and limb development in these FGF-deficient mice? Briefly explain (2 points). F. Compare and contrast the role of FGF in lung development and limb development, and include in your answer an explanation of how two different tissues – developing lungs and developing limbs – can respond very differently to the same signal molecule (3 points). Exam continues on next page5. Despite its name, the protein Yellow is required to produce dark body pigment in Drosophila. In most species of fruit flies, like D. willistoni (right) Yellow protein (and dark pigmentation) is expressed only in the posterior margins of the six abdominal segments. In D. melanogaster and D. biarmipes, however, the region of Yellow expression is expanded; Yellow protein and dark pigmentation is present throughout the last Page 4 two abdominal segments (segments A5 and A6), an expression pattern that corresponds to expression of the transcription factor Abd-B. Similarly, in D. biarmipes, the region of Yellow expression has expanded to include the distal part of the wing, an expression pattern that corresponds to expression of the transcription factor Distal-less. Based on this information and what you have learned about the evolutionary loss of pelvic spines in freshwater sticklebacks, propose a hypothesis that would explain how the expression of Yellow protein has become expanded in melanogaster and biarmipes, briefly explain your hypothesis, and briefly contrast your hypothesis to what you know about the loss of pelvic spines in freshwater sticklebacks (5 points). 6. Expression of shh (sonic hedgehog) in the ZPA (zone of polarizing activity) of vertebrate limb buds is controlled by the ZRS (ZPA Regulatory Sequence), a cis-regulatory element (CRE or enhancer) upstream of the shh coding region (see figure at right). The table below summarizes the results of experiments on mice in which the ZRS of mice is replaced with the ZRS of another organism: ZRS of mouse replaced with ZRS of: shh expression in mouse limb bud Mouse limb development Coelacanth (lobe-finned fish) Normal Normal Human Normal Normal Python (snake that forms rudimentary limb buds) Weakened Partial limb truncation Cobra (snake that does not form limb buds at all) None Severe limb truncation A. What do these findings suggest about the evolutionary developmental basis of limb loss in snakes, and how do these findings relate to what is known about the evolutionary loss of pelvic spines in freshwater sticklebacks? Briefly explain (2 points). B. The ZRS of mice includes multiple binding sites for at least five different transcription factors (HAND2, ETS1, HOXD9, HOXD10, and HOXD13) and is activated when bound to these transcription factors. Based on this observation and the findings presented in the table above, what prediction could you make about the ZRS of pythons compared to the ZRS of mice and the ZRS of cobras? Briefly explain (3 points).