Abstract: The zebrafish Danio rerio is very useful for the study of cell proliferation in the brain. not only because it has been entirely genetically sequenced, but also because teleosts have remarkable abilities to regenerate parts of their central nervous system aster iniury. In adult zebrafish, neurogenesis has been documented predominantly in the cerebellum. and in ventricular zones, especially in the telencephalic ventricle in the forebrain. Neurogenesis in the ventricular zones implies that the cells are turning over and remaining progenitor cells; however this study examines whether these cells are differentiating. Discovering a path of differentiation would lend insight into the regulation of regeneration. This study focuses on possible migration of progenitor cells from the periventricular zone to the optic tectum in zebrafish embryos as they develop. using BrdU to label dividing cells and confocal microscopy to image these cells. The results reveal a smattering of nuclei throughout the optic tectum and encephalon. suggesting migration outward from ventricular areas. Introduction: The study of neurogenesis in the zebrafish brain is an exciting field due to its potential applications for regeneration in the adult human brain, in recovery from neuro¬ degenerative diseases such as Alzheimer's, Parkinson's, and strokes. Neurogenesis in the adult brain has been documented in rodents, in the olfactory bulb and denate gyrus, and in birds, reptiles, and frogs, however the study of brain cell proliferation in teleosts and specifically zebrafish is an emerging field that holds great potential for new discovery. Not only do teleosts have a remarkable ability to regenerate their CNS after injury, but they also have indeterminate growth, so they have no maximum body size and are continually generating new cells. Zebrafish are an ideal experimental model because the zebrafish genome has been entirely sequenced, and because thousands of types of mutants are available for study. Generally, proliferation in teleost brains occurs in the ventricular areas and in the cerebellum, although in the zebrafish brain in particular, neurogenesis has been documented in the olfactory bulb (Byrd and Brunjes, 2001) and in the non-ventricular zones of the forebrain (Mueller and Wulliman, 2002). Proliferation has also been documented in the periventricular zone (Tulloch, 2002, Webster, 2002). However, few studies have explored the fate of these proliferating cells in the periventricular zone. This study focuses on the periventricular zone to determine whether cells that are proliferating in the periventricular zone are migrating outwards into the optic tectum. Proliferating cells in the PVZ could be simply generating cerebro-spinal fluid and turning over. However, this study hypothesizes that some of the proliferating cells are progenitor cells that differentiate into neurons and astrocytic glia in the optic tectum. The results of the study show outlying cells in the tectal and telencephalic zones that imply migration from the PVZ and therefore possible differentiation. Materials and Methods: BrdU Pulse: Three pairs of size-matched juvenile zebrafish were placed in a five-day pulse of bromodeoxyuridine (BrdU), a thymidine analog that stains the nuclei of cells during the S phase of mitosis. 24h after removal from BrdU, the three control fish were fixed. The three experimental fish were fixed at intervals of 10 days, 18days, and 30 days after removal from BrdU. Fixing the fish involved decapitating it and placing the head into 4% PF overnight in the refrigerator, and then placing the head into methanol for an indeterminate amount of time at -20 degrees F. The fish were removed from methanol and rehydrated in 15 minute intervals of 75% methanol, 50% methanol, and 25% methanol, and then water. The brains were then dissected out of the heads and placed in PBS-Tween. Immunohistochemistry: The brains were rinsed in water several times, then rinsed in HCl several times, and then incubated in HCl for one hour. Each rinsing involved a 15 minute soak. The brains were then rinsed in PBS-Tween four times and rinsed in blocking solution four times, and placed in the primary antibody, monoclonal anti-BrdU clone BU-33, in a concentration of 1:100 antibody blocking solution, and incubated overnight in the reffigerator. Asterwards. the brains were rinsed several times in PBS-D-Tween for 15 minutes each and incubated in goat-derived anti-mouse fluorescent secondary antibody, in a ratio of 1:500 antibody. blocking solution. The brains were incubated for 5-6 hours at room temperature. Next the brains were rinsed in PBS-Tween and then sliced into very thin sections and mounted onto slides in glycerol. Confocal microscopy: Digital images were captured of the slides using a confocal microscope. Z-series Tiff- stack movies were compressed into extended views, which were then manually analyzed for the presence of stained nuclei. Results: In the 10-day fish, the control displayed staining in the telencephalic ventricle, while the experimental echoed this staining in the telencephalic ventricle but also revealed outlying nuclei in the forebrain. In the tectal region, the control demonstrated staining in the PVZ and the experimental also revealed outlying cells in the optic tectum. In the 18-day fish, the control did not yield conclusive results in the forebrain. however the experimental indicated several outlying nuclei throughout the forebrain. In the tectal region, the control displayed staining in the central PVZ, whereas the experimental displayed outlying nuclei throughout the center tectal region. The control yielded no conclusive results in either the left or right optic tectum but the experimental displayed unique staining with a smattering of outlying cells in the optic tectum lavers, In the 30-day fish, the control yielded inconclusive results as did most of the experimentat but the experimental displayed staining in the rostral ventricle with a couple of possible outlying cells. Discussion: The BrdU-pulse ensures that the results depict a snapshot of dividing cells, so that it is possible to draw conclusions about the nature of migration. Since all stained cells were stained during the 5-day window of BrdU pulse, the location of the cells after a month can be compared to the final location. All of the controls as well as experimentals display staining in the ventricular zones, which affirms previous research. However the evidence of outlying cells in the optic tectum suggests migration and yields new and intriguing evidence. The outlying cells found in the forebrain do not necessarily suggest migration from the telencephalic ventricle, in light of the evidence discovered by Mueller and Wulliman. because they could have originally been dividing in the forebrain. However, the evidence of stained nuclei in the optic tectum suggests migration because the control only displays staining in the PVZ. Therefore the stained cells in the optic tectum originated in the PYZ and migrated outwards. This migration suggests that cells in the PVZ are progenitor cells that are differentiating into neurons and astrocytic glia and migrating into the optic tectum as they reach their fate. The great variation in data over the time course from 24 hours to one month is most likely not the result of fluctuation in the amount of proliferation, but rather the result of inconsistent staining that prevented some of the slides from revealing useful data. This inconsistent staining and the small sample size of the data make it difficult to draw définitive conclusions about the nature of migration from this study. Howeyer, the evidence of outlying cells, displayed most clearly in the 18-day experimental, vields new and intriguing evidence for the possibility of broader zones of progenitor cells than previously thought, and should be further explored in a greater number of fish over a longer time course. Another future direction for this experiment is to examine the fate of these cells, to use a double-staining procedure with Hu-protein to determine if the outlying cells are neurons or not, and determine zones of neurogenesis as opposed to simply zones of proliferation. In conclusion, the evidence of cells found outlying from the periventicular zone, in the optic tectum, suggests that neurogenesis is occurring in the optic tectum, from progenitor cells in the PVZ. Acknowledgements: 1 would like to thank Christine Byrd for her help with the BrdU protocol, Joe Mayer for his help with the immunohistochemistry, Yoshi Arakaki for his help with the confocal microscope, and Stuart Thompson for his guidance and incredibly precise embryo brain slices. Overview of the Zebrafish brain: 10 a- er ee ee en ce f LV Offactory Bulb Telencephalon Cerebellum Optic Tectum Ventricular Areas: (a. — elencephalic ventricle Periventricular Zone All diagram pictures courtesy of Neuroanatomy of a Zebrafish Brain by Wulliman, Rupp and Reichert. Results: Telence 10-Day Fish 18-Day Fish Experimental: front ventricle: 30-Day Fish Control halic Ventricle: rear ventricle: Experimental: Optic Tectum: 10-Day Fish Control: 18-Day Fish Control: Center Tectal Ventricle Experimental: Experimental Optic Tectum: 18-Day Fish Experimental: lest tectum 30-Day Fish: Rostral Ventricle: Experimental: right tectum 12 Bibliography: Byrd, C. and P. Brunjes, 2001. Neurogenesis in the olfactory bulb of adult zebrafish. Neuroscience. vol. 105, issue 4. pp. 793-801. Mueller, Thomas, and Mario Wulliman, 2003. Anatomy of neurogenesis in the early zebrafish brain Developmental Brain Research. vol. 140. pp 137-155. Uavadia, Ava l, and Elwood Linney, 2003. Windows into development historic, curent. and luture perspectives on transgenic zebrasish. Developmental Biology. vol. 256 isuel pp 1-17. Tulloch, Nathaniel, 2002. In viro Grouth of Zebrafish Neural Precursors. (Unpublished MS. on file at Hopkins Marine Station Library) Webster, Brian, 2002. 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