Developmental Biology

For questions asking you to design an experiment, assume you have access to any reagent or equipment that you want (i.e. don’t worry if something is technically feasible). Focus on describing your experimental approach, the results it will provide, and how you would interpret them. Be as precise and specific as possible!

⦁ (20 points) Loss of limb specification in the snake lineage was an important evolutionary adaptation in their body form. The Kvon et al. (2016) paper identified changes in the ZRS enhancer that likely played a role in this process. In Figure 5, the authors suggest that nucleotides in the binding sites for the ETS1 and homeodomain transcription factors have an increased rate of change in the snake ZRS compared to other species. Devise an experiment that could demonstrate the functional relevance of these transcription factor binding sites in the mouse ZRS for hindlimb development.
⦁ Describe your proposed experiment.
⦁ What is one possible outcome of your experiment?
⦁ Based on the results you describe (b), what conclusion(s) can be drawn from your experiment about the role of the ETS1 and homeodomain transcription factors in limb specification.

⦁ (10 points) What makes one cell type in a developing embryo fundamentally different from another? How do these differences arise during development?

(10 points) This diagram shows somite formation in two species of fish, the platyfish and zebrafish. Even though their embryos are nearly identical in size, the platyfish forms 13 large somites compared to 30 smaller somites in the zebrafish. What specific change to the platyfish somite segmentation “clock and wavefront” might have caused the changes in platyfish somite number and size? Explain how this change would produce the platyfish somite phenotype.

⦁ (15 points) You are working in Dr. Sipe’s lab at Shepherd on newly discovered species of non-vertebrate chordate. This organism does not have a backbone as an adult, but a notochord is present during its larval stage. You think you may have discovered a neural crest-like population of cells in this chordate larva. Based on what you know about NCCs in vertebrates, describe three different experiments you could conduct to determine how similar these cells are to vertebrate NCCs.

⦁ (15 points) The diagrams below show a series of regeneration experiments performed in the newt. In your answers, be as specific as possible in terms of limb structures that will regenerate.

The distal part of the forelimb is amputated, and the remaining limb is inserted into the body wall to heal and establish a blood supply. The tissue boxed in red is subsequently removed from the limb and regeneration is allowed to proceed. What is the outcome of regeneration in limbs a and b?

⦁ Instead of removing the tissue in the red box, you rotate it 90° as shown and implant it at the very proximal end of limb b as shown in the diagram. What is the outcome of regeneration in limb b after this manipulation?

⦁ You perform the same tissue manipulations as in part (b), but you also implant a bead soaked in retinoic acid near the blastema in both limbs. What is the outcome of regeneration in limbs a and b?

⦁ (10 points) Explain how transcription factor expression in a neuron can influence where its axon migrates in the developing embryo.

⦁ (20 points) The schematic diagram shows a cross section through the trunk of a mouse embryo during patterning of the neural tube. The yellow structure is the notochord, and the different populations of neurons produced in the ventral neural tube are shaded blue.

⦁ In the lab you generate a conditional knockout mouse for the patched gene. In this mouse, ONLY the cells of the neural tube lack the patched protein; all other cell types are unaffected. Predict how this genetic manipulation would affect the patterning of ventral neurons in the neural tube (be specific—use the diagram). Why?

⦁ Why must you use a conditional knockout for the experiment described in (a)? Why wouldn’t a complete knockout of the patched gene work?

⦁ Next, you look at neural tube patterning in a mouse completely lacking the gene for gamma-tubulin, a component of the centrosome that is required for cells to make a cilium. Predict what would happen to the patterning of the ventral neurons in the neural tube. Why?

⦁ What other developmental abnormalities would you predict the mice described in (c) might have?