Performing Department
Cell Biology & Molecular Genetics
Non Technical Summary
Spores, pollen and seeds are specialized storage structures where gametophytes or embryos can remain viable during extended periods of quiescence, often in a desiccated state. In response to rehydration or some other signal, the gametophytes or embryos undergo a burst of rapid development and differentiation, either for the production of gametes or for the growth of a new sporophyte. Large quantities of proteins and mRNAs are typically made and packaged into stable complexes prior to entry into quiescence. The control of rapid development involves the utilization of these stored proteins and the translation of these stored mRNAs in a precise spatial and temporal sequence. We are interested in the mechanisms that control rapid development and cell fate determination in the male gametophyte of the semi-aquatic fern, Marsilea vestita. This organism is native to California, and is commonly found at the edges of ponds and vernal pools. It can also be found growing in rice paddies (where it is perceived as a weed). The sporophyte undergoes a gradual drying process at the end of its natural life cycle and produces microspores (and megaspores) that persist in a desiccated state within a hardened sporocarp for many years. Upon rehydration, the microspores enter a precise developmental program involving a series of nine mitotic division cycles to produce 7 sterile cells and 32 spermatids, at precise times and in precise locations inside the spore wall. The spermatids then differentiate into multiciliated spermatozoids that look nothing like any other cell in the organism. We have shown that this process is regulated post-transcriptionally, and relies heavily on the unmasking, processing and translation of many types of stored mRNAs. These mechanisms of emergence from dormancy are highly conserved in widely divergent organisms. In this project, we will focus on how mRNAs are processed and translated in a precise way to facilitate rapid cellular differentiation in a simple and experimentally tractable fern gametophyte.
Animal Health Component
0%
Research Effort Categories
Basic
100%
Applied
0%
Developmental
0%
Goals / Objectives
Rapid development in the male gametophyte of M. vestita is controlled post-transcriptionally. This gametophyte provides a tractable system for studying a variety of basic aspects of developmental regulation. It provides access to mechanisms that regulate how one cell can rapidly divide to produce controlled numbers of two distinctly different cell types.This proposal builds on our previous findings to focus on regulatory mechanisms controlling morphogenesis in a cell that is predisposed for rapid development upon exiting an extended period of quiescence.In this proposal, we present an experimental plan that concentrates on mechanisms that function cooperatively to facilitate rapid, coordinated development in this simple gametophyte. 1) We will complete our characterization of the transcriptome for the developing male gametophyte of Marsilea vestita. 2) We will characterize the roles of different isoforms of kinesins and other cytoskeletal and ciliary proteins in the spermatid maturation. 3) We will analyze the roles of nucleases and proteases in spermatid maturation. These enzymes are upregulated mid-way through gametophyte development.
Project Methods
Our published work includes technologies that we have refined for M. vestita, including in vitro translation assays on isolated RNAs, post-transcriptional silencing by RNAi of developing gametophytes, fluorescent immunolabeling and in situ hybridizations (ISH) for the detection and abundance determinations of proteins and transcripts in fixed cells, and qRT-PCR for RNA abundance with RNA isolates. In unpublished work, we performed RNAseq and constructed a reference transcriptome for the developing gametophyte. We used next-gen sequencing to characterize patterns of RNA unmasking over time of development. We developed technologies to introduce mRNAs encoding fluorescently-tagged fusion proteins into microspores at the time of rehydration. These transcripts are translated and detectable in fixed and living gametophytes with confocal fluorescence imaging. We have developed a series of 'RNA-displacement' assays, which enable us to block the association of sense/antisense complexes at the time of spore rehydration. In addition to finding sense pre-mRNAs that are unique to specific time intervals of development, we have found naturally-occurring anti sense RNAs (NAS-RNAs) that we suspect are important in controlling rates and extents of rapid gametophyte development. The roles played by these NAS-RNAs are going to be the focus of this project.