My broad research interests revolve around the problem of central nervous system (CNS) development and regeneration. Two specific aspects of nervous system development include the initiation of axon growth and proper specification of different neuronal classes. Similarly, the regenerating nervous system requires the re-initiation of axon growth in damaged nerves and/or the replacement of neurons that have undergone cell death in response to damage. I have approached these problems by studying retinal ganglion cells in the developing and regenerating zebrafish. The retina serves not only as an easily accessible model for studying the development and regeneration of CNS in general, but is clinically relevant in its own right due to the potentially blinding diseases and traumas that can affect the retina and the optic nerve. Furthermore, the optical clarity and rapid early development of zebrafish provide an excellent system for visualizing dynamic processes as they occur in the context of a live, developing vertebrate embryo. The specific focus of my research is on the molecular signaling pathways involved in: 1) axon growth regulation and 2) retinal ganglion cell specification from an initial progenitor cell population.

The expression of a number of genes that encode growth-associated proteins (GAPs) accompanies both developmental and regenerative axon growth. This suggests that the same signaling pathways may regulate GAP expression during both modes of axon growth. I tested this theory by asking whether a promoter fragment from a well characterized GAP gene, GAP-43, is sufficient to activate expression in both developing and regenerating neurons. For this purpose, I created stable lines of transgenic zebrafish that express Green Fluorescent Protein (GFP) under the regulation of a 1 kb fragment from the rat GAP-43 gene that contains a number of evolutionarily conserved elements. Analysis of GFP expression in these lines confirmed that the rat 1 kb GAP-43 promoter fragment directs growth-associated expression of the transgene in differentiating neurons that extend long axons. Furthermore, the 1 kb fragment supports developmental down-regulation of transgene expression coincident with neuronal maturation, as is observed with the expression of the endogenous GAP-43 gene. In contrast, this same 1 kb sequence is insufficient for directing GFP expression in regenerating neurons.Together these results show that the developmental regulation of GAP-43 gene expression is evolutionarily conserved between fish and rats. In addition, the signaling elements required for developmental regulation can be maintained, even when those required for activation after injury have been lost or altered in evolution.

I have recently cloned GAP-43 gene regulatory sequences from another teleost species, Fugu rubripes. Fugu genes tend to be more compact (i.e. less "junk" DNA) given that the Fugu genome contains roughly the same number of genes as mammalian genomes but is only 1/8 the size. This feature facilitates the search for specific gene regulatory elements. I am currently undertaking a deletion analysis of the Fugu GAP-43 promoter/enhancer in search of sequences necessary for neuron specific expression of GAP-43 and for regeneration-associated GAP-43 expression in order to identify requirements specific for regenerative axon growth. Future plans include a targeted forward genetic screen utilizing the GAP-43/GFP transgenic fish to identify genes specifically involved in neuronal differentiation and regulation of axon growth.

In addition to studies of axon growth regulation, the GAP-43/GFP fish have proven useful in the study of retinal ganglion cell (RGC) fate determination. This project began with the observation that in GAP-43/GFP transgenic lines, transgene expression in the retina appears first in a subset of undifferentiated retinal progenitors and subsequently localizes to RGCs. This early expression was unexpected since previous reports suggested that GAP-43 expression in the retina is restricted to post-mitotic ganglion cells. However, in situ hybridization revealed that, as with the transgene expression, low levels of endogenous GAP-43 transcripts are observed in retinal progenitors. I am currently studying the transgene expressing progenitors using time-lapse confocal microscopy. Results of these studies indicate that transgene expression in the early retina marks a population of RGC-biased progenitors, thus distinguishing these precursors from an otherwise morphologically uniform set of progenitor cells. I will take advantage of theses lines to investigate early molecular events in neural cell-type specification, and to determine how these early events may affect the generation of subsequent retinal cell classes. In vertebrates, seven major neural classes are specified from a common pool of multipotent progenitors that form the early retina. These seven cell classes differentiate in an ordered progression, RGCs being the first, and occupy distinct positions within the laminar structure of the mature retina. My studies will address questions regarding 1) intrinsic factors involved in commitment to RGC fate, and 2) effects of cell-cell communications between RGC-biased precursors and neighboring cells on subsequent cell specification events.