Harold Smith

Assistant Professor
Ph.D. Microbiology, Columbia University, New York, NY 1994
Center for Advanced Research in Biotechnology
9600 Gudelsky Drive Rockville, MD 20850 USA
Phone: (301) 738-6181
FAX: (301) 738-6255
Email: smithh@umbi.umd.edu

Research: Functional genomics, cell development

We are investigating the mechanisms that govern the development of specialized cells from undifferentiated progenitors. Our model of choice is sperm development, or spermatogenesis, from germ line precursor cells in the nematode Caenorhabditis elegans. This particular system offers several advantages for the study of cellular differentiation. The developing sperm cell progresses through a number of easily observed changes in cell morphology, culminating in the mature crawling spermatazoon. Spermatids can be isolated in bulk for biochemical and in vitro studies. A variety of pharmacological reagents, including calmodulin inhibitors and molecules that alter intracellular pH, can either promote or block some of the latter stages of sperm cell development. C. elegans (colloquially, "the worm") reproduces by either self-fertilization in hermaphrodites or via mating between hermaphrodites and males. This property has been exploited in classical genetic screens to isolate a large number (>60 genes) of spermatogenesis-defective initially called Fer, now Spe) mutants that are self-sterile but cross-fertile. Determination of the complete genome sequence has allowed the generation of DNA microarrays that contain essentially every gene. In conjunction with Sam Ward, Stuart Kim, and Valerie Reinke, we have compared gene expression profiles of worms that make only sperm to worms that make only oocytes, and identified ~1400 genes with elevated expression during spermatogenesis. Current efforts are concentrated on the program of transcriptional regulation for these sperm-enriched genes.

Our goal is to identify the transcription factors that directly control expression of distinct subsets of sperm genes, and to determine the specific stage or process in sperm cell development that is dependent on those genes. We have identified a number of putative binding sites for sperm transcriptional regulators by screening the promoter regions of sperm-enriched genes for over-represented DNA sequence elements. Screening via the yeast one-hybrid system allows the isolation of the relevant binding factor for each of these sequence elements. We have inserted the same elements into the promoters of green fluorescent protein (GFP) reporter transgenes; microinjection of the GFP reporter into C. elegans reveals the in vivo role of these binding sites in sperm-specific gene expression. Several of the sperm-enriched genes identified by microarray are predicted to encode transcription factors; we have begun to obtain deletion mutations in these genes through a PCR-based reverse genetic screen. The mutations will demonstrate the functional role of the transcription factors in sperm development, and, via microarray screening, identify their target genes. By combining the computational, functional, and transcriptional analyses, we hope to determine the developmental program that controls sperm cell differentiation in C. elegans.