Marsilea vestita is a water fern that makes motile spermatozoids. The unique biology of that process has been exploited by the Wolniak lab to develop a uniquely tractable model system for understanding developmental processes and organelle biogenesis de novo. This renewal project will continue these efforts, as well as shed light on aspects of the regulation of gene expression at the messenger RNA level.

Spermiogenesis is a rapid, synchronous process in populations of microspores, initiated by placing the spores into water. Each gamete possesses ~140 cilia, and a distinctive feature of gamete formation in this organism is the de novo formation of basal bodies in cells that lack preexisting centrioles. In each spermatocyte, a discrete particle known as a blepharoplast forms approximately 4 h after spores are placed into water, and during the next two hours the blepharoplast splits and functions as a centrosome for the last mitotic division in the gametophyte. Then, each blepharoplast matures to produce ~140 basal bodies. Dry microspores contain large quantities of stored protein and stored mRNA, and development in the gametophyte is controlled entirely at a post-transcriptional level (no new mRNA is produced). In this project, Dr. Wolniak will focus on four aspects of spermiogenesis in M. vestita that will extend his ongoing work and utilize probes and procedures that he has developed during the past several years.

1) Through RNAi experiments, the Wolniak lab has shown that the translation of specific mRNAs at particular stages of spermiogenesis is necessary for gamete maturation, including formation of the blepharoplast and basal bodies. The first aim of this project tests whether related gene products are translated at the same stage of spermiogenesis. 2) Mago nashi is a highly conserved polarity gene that functions during embryogenesis in a variety of organisms. It is present and essential for proper control of the plane of cell division in the gametophyte. The second aim of this project is to document the patterns of altered division planes after RNAi-induced elimination of Mv-mago, the mago nashi homolog in M. vestita. 3) Cell division planes are tightly linked to cell fate determination in the gametophyte, where one cell in the microspore undergoes successive divisions to produce two cell types, spermatogenous cells and sterile cells. Beyond division control, Mv-mago is also essential for the localization of specific mRNAs into cytoplasmic domains in the microspore that later become the spermatogenous cells. The Mv-mago protein contains two motifs that are identical with those in all other mago nashi proteins. These domains will be added to microspores as epitope-tagged peptides, to block specific functions of endogenous Mv-mago protein (in a loss of function through competition between the peptide and endogenous protein for interacting components), or to rescue gametophytes that have been depleted of Mv-mago mRNA by RNAi treatment. The third aim of this project tests how and when highly-conserved domains in the Mv-mago protein contribute to the control of the plane of cell division and mRNA localization patterns. 4) The Wolniak lab has previously shown that centrin translation is essential for mitotic spindle function and blepharoplast formation in these gametophytes. Epitope-tagged peptides that represent conserved motifs from MvCen1 (the centrin homolog in M. vestita) will be added, to see how and when the centrin peptide disrupts spindle function or blepharoplast formation in normal cells. Then, longer versions of the peptides will be added to RNAi-treated gametophytes, in experiments designed to rescue the loss of function brought about by the RNAi-induced null phenocopy. The fourth aim of this project tests how and when the conserved domains in the MvCen1 protein contribute to spindle function and blepharoplast formation.

The broader impact of this project will be manifested through the training of students. The grant budget includes funds to support a postdoctoral trainee, a graduate student and an undergraduate. These individuals will be trained in a laboratory where undergraduates and graduate students work and learn together, as they perform a variety of experiments on a thoroughly tractable experimental system, to address basic questions about mechanisms controlling cell fate determination and gamete differentiation.

Agency
National Science Foundation (NSF)
Institute
Division of Molecular and Cellular Biosciences (MCB)
Application #
0234423
Program Officer
Elizabeth S. Sztul
Project Start
Project End
Budget Start
2003-05-15
Budget End
2008-04-30
Support Year
Fiscal Year
2002
Total Cost
$575,000
Indirect Cost
Name
University of Maryland College Park
Department
Type
DUNS #
City
College Park
State
MD
Country
United States
Zip Code
20742