Harvard University - Department of Molecular & Cellular Biology

FOUR HOOPES FOR MCB

by Cathryn Delude

June 3rd, 2009

(L-R) Albert Li, Alana Mendelsohn, Sara Trowbridge, and Brady Weissbound

Four undergraduates - Brandon Weissbourd in Catherine Dulac's lab, Alana Mendelsohn in Jeff Lichtman's lab, Sara Trowbridge in John Dowling's lab, and Albert Li in Rachelle Gaudet's lab - received the prestigious Hoopes Prize for their research projects in MCB laboratories. The Hoopes Prize for Excellence in the Work of Undergraduates was established in 1982 from the estate of Thomas Temple Hoopes, 1919, to “grant awards to undergraduates on the basis of outstanding scholarly work or research.” Each of this year's 83 winners will receive a cash award of $3,500.

Brandon Weissbourd Examines Gene Imprinting in Mother-Father Conflict

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(L-R) Chris Gregg, Brady Weissbound, and Catherine Dulac

Brandon (Brady) Weissbourd conducted research in Catherine Dulac's lab on imprinted genes: genes that are differentially expressed depending on the parent from which they were inherited.

This phenomenon is thought to have evolved because in most mammals (and also flowering plants) mothers and fathers are unrelated, and males often mate with multiple females in and across mating seasons. Therefore, mothers are equally related to each of their current and future offspring, while fathers care little for the future reproductive success of mothers, and may even share paternity within the same litter. This discrepancy results in an asymmetry in relatedness between paternal and maternal lineages, resulting in a conflict of genetic interest.

“Maternally inherited genes prefer an optimum level of investment in any given offspring that takes into account future reproductive success, and the success of that offspring's maternal siblings,” explains Brady. “But fathers favor more maternal investment in offspring carrying their genes than what is desirable for mothers” - like having babies that become so large in utero that it is detrimental to the mother's future fertility, or having babies nurse more often, for longer, and to wean at a later period.

This conflict of interests may extend to complex social interactions depending on patterns of relatedness and dispersal. Genomic imprinting has been well characterized in embryonic growth and development, yet the ways in which imprinted genes act upon the brain is poorly understood.

“I was interested in learning more about this phenomenon in the brain because imprinted genes could be the cause of a number of the most common neurological disorders, including autism and schizophrenia,” said Brady.

Catherine Dulac said, “The experimental and intellectual strategy pursued by Brady revealed for the first time how imprinted genes might influence brain function and animal behavior. His tireless efforts translated into exceptional insights as he carried out complex statistical analyses and rigorous literature searches to generate a comprehensive picture of the neural systems targeted by imprinted genes in the brain. Brady's analysis revealed that the parts of the brain that regulate social and motivated behaviors are hotspots for expression of imprinted genes. This provided, for the very first time, a major insight into what imprinted genes do in the brain and how the field should proceed to study imprinting in brain function and behavior.”

Working with a post-doctoral researcher in the Dulac lab, Chris Gregg, Brady mapped the expression of every known imprinted gene to 117 discrete anatomical regions of the mouse brain. They also examined each input and output of those regions as well as their functional connectivity. Once they identified these circuits, they examined imprinting at the cellular level by identifying specific cell-types and systems that may be targeted by imprinted genes.

“So we've attained a global picture of how imprinted genes are expressed in the brain, and have identified regions and circuits that are hotspots for imprinted gene expression,” Brady summarized. He hopes that carefully reviewing these regions and circuits will provide insight into the subtle influence of genomic conflict in neural development, disease, and the formation of adult cognition and behavior.

“Brady's efforts have lead to an outstanding thesis that is already serving as a major resource for me and the lab,” Dulac said. “I am delighted that his scientific contributions have been recognized by the Hoopes Prize.”

After graduating, Brady plans to explore research in immunology or virology - and play some professional beach volleyball - before applying to PhD programs.

Alana Mendelsohn Characterizes Premature Axon Branching in Early Development

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Alana Mendelsohn and Jeff Lichtman

Alana Mendelsohn's research project in Jeff Lichtman's lab revealed new insights into how post-natal neural circuits are refined during early development in the peripheral nervous system. Lichtman uses optical imaging techniques to visualize synaptic rearrangements directly in living animals.

Alana's work was related to his lab's focus on the pattern of motor axon branching within a muscle and its refinement through synapse elimination during postnatal mammalian development. In particular, she was interested in the small number of motor axons that branch prematurely within the nerve fascicle before reaching the target muscle. She wanted to learn more about this still poorly understood extramuscular premature branching and its role in neuromuscular system development.

"Alana asked an important but until now unexplored question concerning the pattern of nerve projections in young animals. Her results were quite interesting because they revealed a degree of extra nerve branching that we had not expected," said Lichtman.

Within muscles, synapses that are eliminated form retraction bulbs and retract back to the previous branch point. That axon branch is said to be refined. "For my research, I did not look at what was happening in the muscle, but rather at the axons that had branched prematurely during development," Alana said.

To characterize the phenomenon of premature axon branching in postnatal development, Alana used transgenic mice that express fluorescent protein in neurons in order to image motor nerves at high resolution. By examining motor nerves in cross section at numerous age points, she found that premature branching is more prevalent at early developmental ages than in adulthood. The younger mice had more premature branches within the nerve and thus more axons entering the muscle.

Alana also noticed that rather than being clustered close to the target muscle, the premature branches are distributed roughly evenly along the length of the nerve. She also observed retraction bulbs - the structures that characterize axons that are in the process of retracting - within the nerve, suggesting that the reduction of premature branching during development is due to refinement along the length of the nerve.

“When I saw that the premature branches end in retraction bulbs within the nerve, I concluded that these were axons that were in the process of being refined. Axonal refinement was previously known to occur within the muscle. My research shows that it also occurs outside of the muscle. So we think that the process of competitive synapse elimination resulting in refinement takes place at a larger scale than previously thought.”

She proposes that the premature branching, while creating non-optimal wiring diagram, may be a byproduct of mammalian nervous system development.

"I am sure we will follow up on her efforts," Lichtman said. "I was really pleased with how much she learned about imaging and the good use she put to this knowledge. I am sad to see her go!”

Alana will begin an MD/PhD program at Columbia University this fall.

Sara Trowbridge Looks at Retinal Development in Zebrafish

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Sara Trowbridge

Professor John Dowling said that Sara Trowbridge worked exceptionally independently on her research in his lab. “The postdoctoral fellow with whom she started the research left Harvard to assume a faculty position elsewhere. The thesis was beautifully written and illustrated, and that she did so much of it on her own is testament to her perseverance and brilliance.”

Sara's research focused on gene expression in the zebrafish retinal pigment epithelium (RPE), which is an accessible part of the central nervous system that is providing clues about neural mechanisms throughout the brain.

The RPE is a pigmented monolayer of cells lying behind the retina that is essential for proper eye functioning and development. Defects in it can lead to retinal degeneration and blindness.

Despite the importance of the RPE, researchers knew little about its interactions with the retina during early development, such as normal retinal lamination and differentiation. Just two years ago, Dowling's lab published the first genomic study of early RPE development. They identified 74 genes that are differentially expressed in the zebrafish RPE at 52 hours post-fertilization (hpf).

Continuing this work, Sara selected seven of these genes for further study based on their potential significance in retinal and RPE development. Using In situ hybridizations, she discovered that five of these seven genes revealed robust expression at 52 hpf in the RPE. She then focused on the gene silverb (silvb) because of its strong and specific expression in the RPE. Also, previous research had linked the Silver protein to retinal development.

“We performed additional in situ hybridizations on silvb and saw that it was also expressed early in development, at 24 and 36 hours after fertilization,” Sara explains.

She decided to perturb its expression to see the effect on the development of the RPE, using injections of antisense oligonucleotide morpholino (MO) at the one-cell stage. The injected embryos lacked pigmentation in the ventral RPE, a phenotype first observed at 24 hpf. “When we analyzed the histology of the injected embryos, we also found that reduced retinal cell density at 36 hours post fertilization and perturbed retinal lamination at 60 hpf.”

By 72 hpf, the injected embryos recovered the retinal lamination, although they still had reduced cell density and eye size.

“So we showed that the silvb gene is expressed exclusively in the RPE during early eye development, and that perturbing it delays retinal lamination and differentiation,” Sara concluded.

Dowling said that Sara's results demonstrate the importance of an RPE-specific gene in proper retinal development, with implications for the understanding of retina-RPE developmental interactions. “Sara was also a delight to have in the laboratory, and we will miss her as she starts a new phase of her life, which next year will be in Paris!”

Albert Li's Structural Studies of TRPV1 Activation by Capsaicin and Acids

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(L-R) Chris Phelps, Albert Li, and Rachelle Gaudet

Albert Li came to Rachelle Gaudet's lab to study a hot topic - the molecular explanation for how certain substances register their sensation of heat on the tongue or skin. Gaudet studies the family of TRP (“trip") receptors to understand nociception, the perception of pain. She uses X-ray crystallography to discover the structure of this family of receptors, which are actually ion channels that open to admit calcium into the cell when tripped by a ligand or by certain temperature ranges.

Li focused on one of these ion channels, transient receptor potential vanilloid receptor 1 (TRPV1). This channel is activated by heat, acidity, and ligands such as capsaicin, the active compound in hot chili peppers. Consequently, it is a target for the study of thermosensation and pain, as well as the development of analgesics.

He wanted to further our understanding of the structure of TRPV1, which will lend some insights into how it functions as a 'molecular receptor for pain.' He focused on studying one means of activating TRPV1, capsaicin binding, and seeing whether he could identify residues important for activation of the channel by capsaicin.

“For the last two years, Albert has spear-headed our effort to construct a mutant TRPV1 protein as a tool for biochemical and biophysical studies of the protein's structure and function,” Gaudet said. “He also performed the first experiments using this tool to obtain structural information about the capsaicin-binding site. It's been a challenging project, where we had to develop or adapt protocols at every step.”

The first part of Albert's project was to engineer a mutant of TRPV1 that would still be functional, that is, would still respond to capsaicin, acidity and heat in a normal way, but is essentially chemically inert. One way to do that is by removing all cysteines, the most chemically reactive amino acid, and replacing them with other amino acids. He could then add back a cysteine and chemically tag it (for example with a fluorophore), knowing its exact location.

Albert succeeded in engineering a cysteine-less TRPV1 that was functionally and structurally similar to wild type TRPV1, based on electrophysiological studies of the full channel and crystallographic studies of the cytosolic ankyrin repeat domain.

A cysteine-less (yet functional) TRPV1 is a useful "scaffold" for mutagenesis experiments, Albert said. “If a mutation changes TRPV1's behavior, I can attribute this change in function to the mutation I made. By working off a cysteine-less channel, I can take this one step further--if I mutate a residue of interest to a cysteine, I know that that is the only cysteine in the entire channel. If I apply one such [chemical tagging] reagent, any change in behavior I see can be attributed to the modification of the sole cysteine in the channel by this reagent.”

He constructed 48 such mutants, each introducing just one cysteine in the putative capsaicin binding site. He wanted to test the sensitivity of these mutants to low pH and capsaicin, so he developed a high-throughput calcium influx assay using a fluorescent radiometric calcium indicator to screen their sensitivities.

By comparing the response to acid and capsaicin, he could tell whether the mutation selectively abrogated TRPV1's capsaicin sensitivity or also abrogated ability to respond to acid, which would imply that the mutation affected more than just the channel's ability to respond to capsaicin. This screen revealed that 22 of the 48 mutations in the putative CBS adversely affected capsaicin sensitivity and 29 mutations adversely affected pH sensitivity - making them less sensitive.

“Albert's results are very intriguing,” Gaudet said. “It's much easier to disrupt the TRPV1 channel activity than I had anticipated. So Albert spent the past semester doing experiments that are helping us prioritize his results so that we can get a better sense of what we need to do to finish the study. But another important impact from Albert's research are the cysteine-less TRPV1 protein and the methods and protocols that he developed, which will be used for years to come for our studies of TRPV1 and related ion channels.”

In the fall, Albert will enter an MD program at the New York University School of Medicine.