Cellular Neurobiology BIPN 140 Fall 2016 Problem Set #8

Transcription

1 Cellular Neurobiology BIPN 140 Fall 2016 Problem Set #8 1. Inductive signaling is a hallmark of vertebrate and mammalian development. In early neural development, there are multiple signaling pathways involved in this process. a) What is the general mechanism by which inductive signals act during induction? An inductive signal/ligand that is either secreted or cell attached binds to the corresponding receptor, which then signals to the nucleus through binding to DNA, and ultimately alters transcription. b) Among the inductive ligands Nick presented in lecture, the bone morphogenetic proteins (BMPs) have different functions in the neural plate than those they have elsewhere. What do BMPs promote in mesodermal cells (outside of ectodermal cells/neural plate)? Describe the mechanism by which this is achieved. BMPs promote bone formation outside of the neural plate. These secreted proteins bind to surface serine receptor kinases that phosphorylate the SMAD proteins, which then translocate to the nucleus in order to bind to DNA and alter transcription. c) How is the mechanism of action of BMPs in b) avoided to achieve what they do in the neural plate? Provide the name of the proteins which assisted in the modulation of the signaling event. BMPs are antagonized in the neural plate by secreted proteins such as noggin and chordin. Since BMPs are blocked from their receptors, the neural plate continues along a path of neuralization which block bone (or epidermal) production but not neuronal induction. d) In the absence of sonic hedgehog (Shh), an inhibitory protein complex modulates a family of transcriptional regulators (such as Gli1). Describe the outcome of inductive signaling in the presence of Shh. Transduction of signals via Shh requires cooperative binding to Patched and Smoothened coreceptors (both are surface receptor proteins). This causes disassembly of the inhibitory complex and allows Gli1 to be translocated into the nucleus, where it positively regulates the expression of genes that establish neural identity. 2. Formation of the nervous system: gastrulation and neurulation. a) Which process is critical for the initiation of neural development? Why? Gastrulation. It defines the midline as well as the anterior-posterior and dorsal-ventral axes of all vertebrate embryos. b) Gastrulation begins as the local invagination of a subset of cells in the very early embryo. By the time invagination is complete, what three germ layers is the embryo made out of? Outer ectoderm 1

2 Middle mesoderm Inner endoderm c) Which layer gives rise to the entire nervous system? Neuroectoderm, which is the ectoderm layer that is directly above the notochord. d) How is neurulation initiated? The notochord sends inductive signals to the overlying ectoderm that causes a subset of cells to differentiate into neuroectodermal precursor cells. This layer may be now referred to as the neural plate. e) Fill in the blanks: After the neural plate becomes defined in early neurulation, it begins to fold at the midline forming the neural groove. The neural plate immediately above the notochord differentiates into the floorplate, whereas the neural crest emerges at the lateral margins of the neural plate. Once the edges of the neural plate meet in the midline, the neural tube is complete. The mesoderm adjacent to the tube then thickens and subdivides into structures called somites (precursors of the axial musculature and skeleton). As development continues, the neural tube adjacent to the somites becomes the rudimentary spinal cord, and the neural crest gives rise to the sensory and autonomic ganglia. The anterior ends of the neural plate grow together to the midline and continue to expand, eventually giving rise to the brain. 3. To study neurogenesis, bromodeoxyuridine (BrdU) is injected into a pregnant mouse at the beginning of neural migration in developing pups (experiment 1). In another mouse, BrdU is injected 12 hours later in development (experiment 2). The pups are born, sacrificed at postnatal day 25, and brains are collected and stained for BrdU. A band of labeled neurons is visible in both experiments in the cortex and cerebellum. a) What determines the birthdate of a neuron? A neuron s birthdate is the time of final DNA synthesis. b) How does BrdU act as a label for cell birthdate? BrdU is incorporated into DNA during DNA synthesis, which occurs during mitosis. During asymmetrical division, a neural stem cell divides, producing one neural stem cell and one neuroblast. The neuroblast is post-mitotic, so it will only be labelled if it was generated in the presence of BrdU. c) In the cortex, would you expect the band to be closer to the inside or outside in experiment 1 as compared to experiment 2? Closer to the inside, because the cortex develops from inside to outside. d) In the cerebellum, would you expect the band to be closer to the inside or outside in experiment 1 as compared to experiment 2? Closer to the outside, because the cerebellum develops from outside to inside. 2

3 4. Many pathfinding mechanisms contribute to the guidance of axons to their targets during development. These mechanisms usually consist of ligands to receptors that ultimately alter the neuron s transcription of specific genes. a) What are the two classes of diffusible molecules that affect pathfinding and what are their effects? Chemoattractants attract the axon to migrate into a certain direction (axon moves towards chemoattractants), while chemorepellents repel migration in a certain direction (axon moves away from chemorepellents). b) Predict how the following scenarios would influence axonal migration at the midline of the nervous system. Assume that the receptors are functional, if not specified otherwise. i) Axon in the proximity of the midline that expresses Slits and Semaphorins. The axon does not migrate to the midline due to the lack of Netrin- a chemoattractant that guides the axon to the midline. Both Slits and Semaphorins act as chemorepellents that are able to bind to activated receptors on the axon only at the midline itself. Therefore, the axons should experience no migration toward the midline. ii) Axon in the proximity of the midline that expresses Netrin and conformationally modified Slit as a result of a rare genetic mutation. The axon migrates to the midline upon the binding of Netrin to DCC receptors. However, due to the conformational change, Slits are unable to bind to the Robo receptors. Therefore, one would expect that the axon would stay at the midline and never cross it. iii) Robo receptor knock-out axon near the midline that expresses Netrin and Semaphorins. The axon migrates to the midline due to the presence of Netrin binding to its DCC receptors. Once it reaches the midline Semaphorin will be able to bind to activated plexin and neuropilin receptors which will repel the axon from the midline. Therefore, the axon will experience normal crossing of the midline, despite lacking Robo receptors (binds Slits). 5. Axon guidance during neural development allows neurons to accurately reach their targets by following precise paths in the nervous system. a) What were the 3 relatively simple animal models discussed in lecture? Zebrafish embryo, grasshopper embryo and the nematode embryo (C. elegans). b) As grasshopper limbs develop, distal sensory neurons are the first to grow toward the CNS, before axons from other neurons fasciculate with these pioneer axons. You decide to do a laser ablation to remove some of the guidepost cells near the developing limb bud before the pioneer cells arise. How will this affect the pathfinding of their followers? What if the ablation is done after pioneer cells arise? Since the pioneers need to extend their axons to nearby guidepost cells, which provide routes to reach the CNS, removal of these landmarks would disturb pathfinding for newly formed pioneers at the site of removal. Pioneers may wander around in the limb and never reach the CNS. However, other cells can acquire pioneer cell fate away from the site, but near intact guidepost cells, and continue the pathfinding process. In both ablation scenarios, followers would still be able to fasciculate with pioneer axons and extend their bundled axons into the CNS. 3

4 c) In some cases of axon pathfinding in a more complex model, such as frog embryos, a topographic map arises during development for the correct establishment of axonal projections. What is a topographic map? Neighboring neurons at one location project axons to adjacent neurons in another location. d) Explain how the gradients of cell surface molecules facilitate the correct projections of retinal ganglion cells in the optic tectum. Why does the lock and key hypothesis appear untenable? In the eye and the tectum, ephrins and Eph receptors are distributed in complementary gradients so that similar levels of ligand and receptor are matched. As the retinal ganglion cells extend their growth cones containing the Eph receptors onto the tectal cells (which express the ephrin ligands), growth cones with low concentration of Eph receptors would migrate to the high end of ephrin gradient, while the ones with high concentration of EphR migrate to the low end of the ephrin gradient. On the other hand, the lock-and-key model states that each retinal ganglion cell has a chemical label (such as a specific receptor) that is complementary to another chemical label (such as a ligand for this receptor). However, the large discrepancy between the number of proteins available to serve as labels and the number of connections that have to be made led to the conclusion that this hypothesis cannot be supported. 6. a) When determining whether neurons are capable of neurotransmitter switching, why is it important to find out whether any neurogenesis or apoptosis has occurred? Birth or death of neurons could indicate a different mechanism by which the proportion of neurons expressing a given neurotransmitter is altered. If birth or death occurs, it is possible that newly born neurons express the new neurotransmitter, and those expressing the original neurotransmitter have died. b) How is the mechanism of initial neurotransmitter specification different from later mechanisms of specification? Initial specification is regulated by genetic programs, whereas later mechanisms are activitydependent. c) Why is the way activity regulates the proportion of glutamatergic vs. GABAergic neurons considered homeostatic? Low activity promotes neurons to make glutamate, which is an excitatory neurotransmitter. This will increase the amount of activity. High activity promotes neurons to make GABA, which is typically an inhibitory neurotransmitter. This will decrease the amount of activity. In this way, the balance of activity is maintained by tuning the number of glutamatergic and GABAergic neurons. d) What are reserve pool neurons? Why might it be an advantageous strategy to rely on reserve pool neurons, rather than relying on neurogenesis? Reserve pool neurons are neurons that are already wired into circuits, already make and release neurotransmitters and have one set of functions. However with appropriate stimulation they can respecify (switch) their neurotransmitters and acquire another set of functions. 4

5 In a reserve pool neurons are already in place and connected in the circuit. In the adult brain, to tune a circuit using neurogenesis, new neurons would need to migrate (perhaps over large distances) and also establish new connections. This would be mechanistically challenging, as guidance molecules established during development are not always maintained. Having a reserve pool also allows for faster flexibility, as switching can occur fairly rapidly (in the example of the rat photoperiod, switching occurred after a week). If switching regulates a behavior that is advantageous to survival, than the ability to respond rapidly would increase survival. e) What are phenotypic checkpoints? What examples can you think of? Phenotypic checkpoints are intermediate differentiated states that need to be expressed during development for expression of the next intermediate differentiated state (on the way to acquisition of the terminal differentiated state). Expression of calcium-dependent action potentials and arrival at the midline by growth cones are two examples. The first enables transmitter switching and the second enables growth cone crossing of the midline. 5