Here, we show that the polyamine spermidine plays a key role as a morphogenetic determinant during spermatid development in the water fern Marsilea vestita. Spermidine levels rise first in sterile jacket cells and then increase dramatically in spermatogenous cells as the spermatids mature. RNA interference and drug treatments were employed to deplete spermidine in the gametophyte at different stages of gametogenesis. Development in spermidine-depleted gametophytes was arrested before the completion of the last round of cell divisions. In spermidine-depleted spermatogenous cells, chromatin failed to condense properly, basal body positioning was altered, and the microtubule ribbon was in disarray. When cyclohexylamine, a spermidine synthase (SPDS) inhibitor, was added at the start of spermatid differentiation, the spermatid nuclei remained round, centrin failed to localize into basal bodies, thus blocking basal body formation, and the microtubule ribbon was completely abolished. In untreated gametophytes, spermidine made in the jacket cells moves into the spermatids, where it is involved in the unmasking of stored SPDS mRNAs, leading to substantial spermidine synthesis in the spermatids. We found that treating spores directly with spermidine or other polyamines was sufficient to unmask a variety of stored mRNAs in gametophytes and arrest development. Differences in patterns of transcript distribution after these treatments suggest that specific transcripts reside in different locations in the dry spore; these differences may be linked to the timing of unmasking and translation for that mRNA during development.

In Caenorhabditis elegans, P granules are germline-specific, RNA-containing granules that segregate into the germline precursor cell during early embryogenesis. In this short report, PAN-1, which previously has been found by others in screens for genes causing larval molting defects, is identified here as a novel P-granule component and a binding partner of GLH-1 (Germline RNA Helicase-1), a constitutive, germline-specific, P-granule protein. The PAN-1 predicted protein contains multiple leucine-rich repeats (LRRs) and regions with similarities to F-box proteins. Antibodies raised against PAN-1 reveal it is present both in the soma and the germline. In the germline, PAN-1 uniquely localizes to P granules from the first larval stage onward and is unusual for a P-granule component in lacking recognizable RNA binding motifs. Homozygous pan-1(gk142) deletion worms arrest as larvae that are unable to molt and this phenotype is also seen with pan-1(RNAi) into wild type worms. pan-1(RNAi) into the somatic RNAi-defective strain rrf-1(pk1417) bypasses the larval arrest and allows an assessment of PAN-1 function in the germline. We find pan-1(RNAi) is variably effective in knocking down PAN-1 protein and results in adult progeny that display multiple germline defects. These phenocopies range from under-proliferation of the germline, as also seen with loss of GLH-1, to the induction of endomitotic replication in oocytes, both defects that result in sterility, to fertile animals with significantly reduced progeny numbers. Thus, while loss of PAN-1 in the soma inhibits molting, this report demonstrates that PAN-1 is also a P-granule component that is essential for fertility.

The endosporic male gametophyte of the water fern, Marsilea vestita, provides a unique opportunity to study the mechanisms that control cell fate determination during a burst of rapid development. In this review, we show how the spatial and temporal control of development in this simple gametophyte involves several distinct modes of RNA processing that allow the translation of specific mRNAs at distinct stages during gametogenesis. During the early part of development, nine successive cell division cycles occur in precise planes within a closed volume to produce seven sterile cells and 32 spermatids. There is no cell movement in the gametophyte; so, cell position and size within the spore wall define cell fate. After the division cycles have been completed, the spermatids become sites for the de novo formation of basal bodies, for the assembly of a complex cytoskeleton, for nuclear and cell elongation, and for ciliogenesis. In contrast, the adjacent sterile cells exhibit none of these changes. The spermatids differentiate into multiciliated, corkscrew-shaped gametes that resemble no other cells in the entire plant. Development is controlled post-transcriptionally. The transcripts stored in the microspore are released (unmasked) in the gametophyte at different times during development. At the start of these studies, we identified several key mRNAs that undergo translation at specific stages of gametophyte development. We developed RNA silencing protocols that enabled us to block the translation of these proteins and thereby establish their necessity and sufficiency for the completion of specific stages of gametogenesis. In addition, RNAi enabled us to identify additional proteins that are essential for other phases of development. Since the distributions of mRNAs and the proteins they encode are not identical in the gametophyte, transcript processing is apparently important in allowing translation to occur under strict temporal and spatial control. Transcript polyadenylation occurs in the spermatogenous cells in ways that match the translation of specific mRNAs. We have found that the exon junction complex plays key roles in transcript regulation and modifications that underlie cell specification in the gametophyte. We have recently become interested in the mechanisms that control the unmasking of the stored transcripts and have linked the synthesis and redistribution of spermidine in the gametophyte to the control of mRNA release from storage during early development and later to basal body formation, cytoskeletal assembly, and nuclear and cell elongation in the differentiating spermatids.

No abstract