The goal of this project is to elucidate the mechanisms that control cell fate determination in the male gametophyte of the fern Marsilea vestita. Specifically, studies will focus on the spatial and temporal regulation of translation that underlies basal body formation and nuclear remodeling in the spermatids. Spermatogenesis in this fern is a process that is activated by placing dry spores into water and involves a series of nine rapid cell division cycles in precise planes within the spore wall to produce 32 spermatids and 7 sterile cells. There is no movement of cells, so size and position within the spore wall defines cell fate. Gametophyte development takes place in the absence of new transcription; instead, the gametes are formed with proteins that were already present in the spore, or translated from mRNAs that had been transcribed and stored prior to spore desiccation. Within the spermatids, differentiation involves the formation of basal bodies in the absence of preexisting centrioles in a unique particle known as a blepharoplast. The spermatids then form an elaborate signature microtubule cytoskeleton known as the multilayered structure (MLS). The later phases of spermatid maturation involve nuclear remodeling, cell reshaping, and finally ciliogenesis, to produce a multiciliated, spirally-shaped motile gamete that looks nothing like the cell that gave rise to it. None of these events occurs in the sterile cells, though all of the cells present within the spore wall arose from the same progenitor. Thus, the male gametophyte of Marsilea provides a unique opportunity to study multiple aspects of cellular differentiation in populations of synchronously developing cells, where the cells are permeable to the entry of macromolecules at the time the spores are placed into water. The specific objectives of this project are to (1) determine how the core components of the exon junction complex affect patterns of centrin translation essential for the process of de novo basal body formation; (2) characterize the RNA-binding proteins that regulate the abundance of alpha-tubulin, which, together with centrin, affects basal body production and formation of the MLS; and (3) determine how spermidine affects temporal and spatial patterns of translation, thereby influencing cell fate specification within the gametophyte.
This project will provide research training and mentoring of undergraduate and graduate students, postdoctoral associates and visiting scholars. In addition, laboratory courses have been developed that provide training in cell biology and microscopy for undergraduate and graduate students and a website focused on an "Introduction to Mitosis" has been developed and is accessible by the public at www.life.umd.edu/CBMG/faculty/wolniak/wolniakmitosis.html.
