is an aquatic, heterosporous water fern whose sporophyte resembles a four-leaf clover. Its microspores and megaspores are meiotic products that desiccate and become dormant after they are formed. Upon rehydration, the microspores develop rapidly to produce male gametophytes that make multiciliated spermatozoids [for review, [1
Like other rapidly developing systems [2
], male gametophyte development and spermatid differentiation of M. vestita
depends on little or no new transcription. The microspore becomes transcriptionally silent during its desiccation and remains so upon rehydration and initiation of spermatogenesis [8
]. Therefore, transcriptional activity essential for gametophyte development occurs prior to spore desiccation, and after spore hydration, spermiogenesis relies on the translation of stored mRNAs [for review, [1
]. In this system, the mobilization, distribution and processing of stored mRNAs in the gametophyte underlies patterns of rapid development.
Not surprisingly, the translation of specific stored transcripts is under tight temporal and spatial control [9
]. One example of this spatial and temporal regulation of stored transcripts is centrin mRNA. Centrin is a calcium-binding phosphoprotein that has been shown to be essential in motile apparatus formation in the microspore of M. vestita
]. Centrin mRNA is uniformly distributed throughout the cytoplasm of the microspore from the onset of gametophyte development, but centrin protein levels are barely detectable during of the first four hours after the spores are hydrated. Beyond that time point, centrin protein levels increase dramatically, but only in the spermatogenous cells, where they remain elevated through the completion of gamete formation [13
]. Thus, the translational capacity for centrin protein synthesis is asymmetric, because centrin mRNA is present in the cytoplasm of both sterile and spermatogenous cells in the gametophyte, but centrin is translated only in spermatogenous cells [12
]. Centrin RNA was examined in this study because of the extensive amount of preexisting knowledge regarding its spatial and temporal dynamics during microspore development [for review see: [1
]. Similarly, temporal and spatial control over translation has been observed for a number of other transcripts [12
] and proteins [10
] in these gametophytes.
An important mechanism regulating gametophyte development is the unmasking of stored transcripts for translation [for review, [14
]. Within this context we define "masked RNA" as mRNA whose translational state is initially inhibited, but later is "unmasked" to become translationally competent. This pool of masked mRNA is stored in the nucleus of the desiccated spore [15
]. We refer to mRNA that is uniformly distributed in the cytoplasm of all cell types in the gametophyte, but does not appear to be translated at anytime during development as quiescent cytoplasmic mRNA (qc-mRNA).
Recently, we found that the polyamine, spermidine (SPD), acts as a temporal regulator for releasing the masked, stored transcripts in the gametophyte [1
]. Exogenous additions of SPD and other polyamines at the time of spore hydration cause the precocious unmasking of spermidine synthase (SPDS) mRNA in addition to other masked transcripts including centrin, PRP-19, and gamma-tubulin [15
]. High concentrations of SPD also arrest division cycles, presumably because of premature transcript unmasking. Precociously unmasked transcripts display an intriguing pattern of distribution, and appear as distinct particles in the nucleus [15
]. These findings led us to hypothesize that a subset of masked transcripts is stored within the nucleus of the microspore and that the temporal regulation of these transcripts is dependent on unmasking as a prerequisite for translation essential to the proper completion of spermatogenesis. Since masked transcripts appear to be stored within the nucleoplasm of the microspore as it undergoes desiccation, we were also interested if these masked transcripts are associated with known nuclear bodies.
Nuclear speckles are small aggregations (~1 μm) of 20-25 nm granules that occupy the interchromatin space of many eukaryotic nuclei [16
]. Several types of pre-mRNA processing proteins are constituents of nuclear speckles [17
], and speckles also contain a subset of poly(A)+ RNA [18
]. Within the interchromatin space of the nucleus, speckles are often localized adjacent to genes with high transcriptional activity [21
]. While a direct role for nuclear speckles in transcription and post-transcriptional modification has neither been confirmed nor disproven definitively, several lines of evidence suggest a role for speckles in the transport and splicing of pre-mRNA [for review, [27
]. Interestingly, upon an inhibition of transcription, speckles have been observed to enlarge and assume a rounded morphology, which has been suggested to result from the storage of pre-mRNA splicing factors [28
]. In addition to pre-mRNA splicing factors, a subset of poly(A)+ remains associated with enlarged nuclear speckles within the nucleus under conditions where transcription has been inhibited [18
]. The purpose of this nuclear retention of RNA is unknown.
Stress conditions such as hypoxia, inhibition of respiration, transcription, phosphorylation and ethanol treatment have been shown to cause the sequestration of the Exon Junction Complex (EJC) core component eIF4A-III to several subnuclear structures including nuclear speckles [31
]. It is likely that mRNA associated with these sequestered components will be retained within the nucleus and not translated. It is likely that the association of core components of the EJC with subnuclear RNA and/or nuclear speckles could play a vital role in regulating a subset of cellular processes. Previously experiments have demonstrated the ubiquitous expression of EJC core component Mago nashi in other plant systems and that the loss of this expression has extensive effects on development [33
]. Since the microspore of M. vestita
is a transcriptionally silent system that relies on the translation of stored mRNA after the spore is released from desiccation, and contains a subset of nuclear localized masked transcripts, we suspected that examining nuclear speckle dynamics might lead to insights into developmental control in the maturing gametophyte.
In this study, we have examined nuclear speckle dynamics during microspore entry into dormancy and transcriptional quiescence. We show that in addition to cytoplasmic stores, aggregated nuclear speckles serve as sites of poly(A)+ RNA storage. We developed a novel variation on fluorescence in situ hybridization (FISH), in an assay designed to distinguish between masked and unmasked (qc-mRNA) populations of the same transcripts in fixed cells. We demonstrate the utility of this assay by tracking the movements of specific transcripts initially stored in association with nuclear speckles into the cytoplasm of the antheridial mother cell. We show subsequent movements of masked transcripts into spermatogenous, but not jacket cells of the developing gametophyte, and that this asymmetry may be regulated via the EJC component Mv-Mago. The asymmetric distribution of one of these transcripts (centrin) mirrors its pattern of translation and presents a likely mechanism for post-transcriptional regulation essential for cell fate determination during the rapid and precise process of spermatogenesis in M. vestita.