Ida Sibylle Haussleiter

Home Institution

Host Institution
Neural Plasticity Research Group Department of Anesthesia and Critical Care, Massachusetts General Hospital and Harvard Medical School
Research Mentor: Clifford Woolf, M.D., Ph.D.
Clinical Rotation: Pain Center, Massachusetts General Hospital

E-Mail: ida@haussleiter.de

Research Topic
see Abstract
Personal Reactions to the U.S. Experience
This was one of the best decisions I have ever made! I thank my family that they pushed me to go. The research level here in America is unbelievable, the possibilities are unlimited, and the people are undeniably great. To spend this year in a world famous laboratory is a once in a lifetime chance for me and definitely influences my work plans for the future. Not only that, but I also found very good friends, as well as two wonderful roommates here in Boston.
Greatest Difficulties Encountered
1. If you want to take a bus, just wait on the street. Once in a while, and definitely not following any logic or chronological rules, a bus shows up. On rainy and snowy days there is no bus at all, of course. Normally you have no idea where the bus comes from and where it goes…. But enjoy the ride.
2. What kind of difficulties can you imagine in a land where people do not know quark, bitter lemon, woodruff jello and gluehwein? And I did not mention real bread, real chocolate and real coffee…
3. Spending two nights in an emergency room is only fun as long as you are on the good side of the game.
Most humorous incident
I was on my way home from the lab, trying to catch a certain bus… I was lucky and saw it standing at the bus stop, but as soon as I started to sprint to catch it, the bus driver took off. Completely disappointed I slowed down and already expected 30 minutes waiting time, when suddenly a police car drove backwards, stopped beside me and a very polite officer asked me if the leaving bus was mine. I nodded, remembering my never-talk-to-strangers-lesson, and earned a very cheering "jump in babe, we'll catch it…!"
Helpful Hints for Future Students
1. Cold means cold. (for all future Bostonians)
2. Stop means stop. (for all future car drivers)

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Abstract on Research Topic
Crouching Rabbit, Hidden DRAGON

Authors
Ida S. Haussleiter, Tarek A. Samad, Jason A. Campagna, and Clifford Woolf

Institution
Neural Plasticity Research Group, Department of Anesthesia and Critical Care, Massachusetts General Hospital and Harvard Medical School, Boston, MA
Background and Purpose
The highly ordered development of neuronal pathways is essential for the establishment of the nervous system. Axonal growth is integral to neuronal development and comprises multiple components: neurite induction and outgrowth, axon guidance, target recognition and innervation.
Several transcription factors have been found to control the connection between primary sensory neurons and their central targets in the spinal cord. In this way, they orchestrate the proper connectivity of the somatosensory system during embryogenesis. The regulation and mechanics of these transcription factors, as well as their interaction with other stimulating or inhibiting mediators within neural development, are complex and the effectors by which they produce their actions during development are unknown.
DRG11 is a transcription factor that is required for the development of nociceptive neurons. It is essential for the normal establishment of those sensory neural pathways that detect noxious stimuli and contribute to the pain. DRG11 is a paired homeodomain transcription factor, which is expressed early in DRG sensory neurons in the embryo (E10.5) as well as in dorsal horn neurons (E12).
Using a mouse genomic library, we have screened for DRG11-regulated target genes to investigate the molecular and cellular mechanisms responsible forof neural development and nociception. We have identified a new gene DRAGON (DRG11 responsive axonal guidance & outgrowth of neurite gene), whose expression is induced by DRG11. DRAGON cDNA is 1311 base pairs long, encoding for a protein of 436 amino acids, and is expressed in primary sensory neurons, the spinal cord and brain. Sequence analysis indicates that DRAGON is a member of a new gene family, which comprises three murine genes encoding for GPI anchored proteins. Members of this family - including DRAGON, DL-M (muscle) and DL-N (neural) -share a sequence homology of 50-60%.
 

Project
We have generated and characterized a polyclonal antibody against DRAGON. My project was designed to produce anti-DL-M and -DL-N polyclonal antibodies.
- Generation of specific polyclonal anti-DL-M / -DL-N antibodies in rabbit: Highly immunogenic peptide sequences of each gene were identified, synthesized and conjugated to a carrier to enhance their antigenic profile. Two peptides were synthesized for each protein, to increase the probability of obtaining a highly reactive polyclonal antibody and two rabbits were used for each peptide. Antibody titer in the rabbit serum was determined by ELISA.
- Recombinant expression in HEK293T cells and testing of the newly obtained antibodies: We subcloned DL-M and DL-N in a mammalian expression vector. Human Embryonic Kidney cells HEK293T were transfected with DL-M- or DL-N-construct. The affinity of the various antisera was tested against transfected cells and compared with non-transfected ones.
- Western Blot and immunohistochemical assays: These assays are being used to evaluate the specificity of the raised antibodies against native protein. They will be performed on different embryonic and adult mouse tissues to assess the relative expression and distribution of DL-M and DL-N proteins in spinal cord, brain and skeletal muscle tissues.
- Knock-Out Mouse: To test whether DRAGON and DL-N are required for the development of the peripheral and the central nervous system, we have started to generatiegene-targeted mice carrying a null mutation in these genes.

Future prospects
After characterization of the expression and distribution of DL-M and DL-N in the mouse, we plan to investigate their function in vitro using neuronal cell culture assays.