Spermatogenesis in the semi-aquatic water fern, Marsilea vestita, is a rapid, synchronous process that produces motile gametes in only 11 h [1]. Similar to other rapidly developing systems, spermatogenesis in male gametophytes of Marsilea is controlled at a post-transcriptional level. Virtually all the RNA required for development is present in the dry spore at the time of its rehydration and almost no additional transcription is needed for spermiogenesis to reach completion [2–4]. Rapid development is dependent on the unmasking of pre-mRNAs [5], which are stored in dry spores within nuclear speckles [6], and then, the processing of these pre-mRNAs for translation [7]. Spermatogenesis begins when microspores are exposed to water. Shortly after hydration, the gametophyte initiates a developmental program that culminates with the production of 32 corkscrew-shaped spermatozoids, each with ~140 cilia [8–11]. Development can be divided into two phases. The first phase consists of a series of nine mitotic division cycles that produce 32 spermatids that are surrounded by seven sterile cells. All divisions are complete approximately 5 h after microspore hydration. During the second phase, only the spermatids undergo drastic morphological changes and differentiate into motile, corkscrew-shaped spermatozoids [4, 12].
This unusual shaping of the gamete is achieved through the elongation and coiling of the nucleus and mitochondria along a coiled ribbon of crosslinked microtubules. Basal bodies, already formed de novo, are placed in two rows at regular intervals along the dorsal face of the microtubule ribbon to become the sites of ciliogenesis [8, 11]. At first, basal bodies are oriented so cilia diverge away from each other and are parallel to the plasma membrane of each spermatid. Near the end of spermiogenesis, the basal bodies rotate 90° so that the ciliary axonemes protrude vertically from the microtubule ribbon and nuclear coil [13]. At 9.5 h of development, an extension of cytoplasm begins to grow around the anterior end of each spermatid and eventually fuses together to surround each cell. This creates an internal, but extracellular space that contains the microtubule ribbon and organelle coil plus all of the cilia. Upon release from the microspore, each spermatozoid breaks free from the surrounding cytoplasmic extension and leaves behind a thin vesicle-like structure [11]. The ciliary axonemes have the typical 9?+?2 architecture [13] found in motile organisms and spermatozoids are able to swim towards the megaspore for fertilization.
We are interested in the processes that regulate spermatid differentiation and ciliogenesis during male gametophyte development in Marsilea. Proteins important for ciliary assembly and function are moved to the distal ends of forming axonemes by intraflagellar transport (IFT) involving members of the kinesin-2 family. Heterotrimeric kinesin-2 consists of a kinesin-2?, a kinesin-2?, and a kinesin associated protein (KAP) that regulates cargo binding [14]. In Chlamydomonas, heterotrimeric kinesin-2 is necessary for IFT [15–18]. Kinesin-2 can also function as a homodimer of two kinesin-2? subunits. Kinesin-2?, also known as OSM-3 or KIF17, has a distinct role in assembling sensory cilia that is separate from heterotrimeric kinesin-2 [19–24]. Although the most common mechanism for axonemal assembly is dependent on IFT function, Plasmodium falciparum and the sperm flagella of Drosophila are able to build motile cilia and flagella using IFT-independent mechanisms. In this case, cilia are assembled in the cytoplasm [25–27]. Ciliogenesis in Marsilea does not occur in this way; instead, cilia are assembled in growing membrane extensions from basal bodies that are positioned along a microtubule and organelle coil. Therefore, we anticipated that ciliogenesis in Marsilea is reliant on IFT-dependent mechanisms of transport and assembly. In support of this idea, we found a variety of transcripts encoding IFT-associated proteins (kinesin-2, dynein-1b, IFT-A, and IFT-B subcomplex proteins) in the assembled transcriptome from the male gametophyte of this organism [7].
Phylogenetic analysis of kinesin-9 shows the existence of two subfamilies, kinesin-9A and kinesin-9B. Kinesin-9A includes KIF9 and kinesin-like protein 1 (KLP1), while kinesin-9B includes the KIF6 protein [28, 29]. Kinesin-9A is localized to the central pair of microtubules in the Chlamydomonas axoneme [30] and is necessary for motility in both Chlamydomonas [31] and Trypanosoma brucei [29]. Kinesin-9A appears to be important for motility by regulating the activity of flagellar dynein [31] and by interacting with Hydin, which is necessary for motility in algae [32], trypanosomes [33], and mice [34]. Less is known about the function of kinesin-9B; however, in T. brucei, KIF9B localizes to basal bodies and the flagellum where it is necessary for the construction of the paraflagellar rod [29].
Although the processes that regulate the structure and function of cilia are highly conserved, not all organisms make ciliated cells. This is most pronounced during the evolution and adaption of land plants from green algae. Conifers and angiosperms never make any ciliated cells, while lower plants known as embryophytes, including the ferns, mosses, liverworts, and certain members of the gymnosperms (e.g., Ginkgo biloba and the cycads) only produce cilia in their male gametes. In conjunction with the loss of cilia in the ‘higher’ organisms, the reduction [35], or complete absence, of proteins important for ciliogenesis and motility is also observed. For example, comparative analyses of the kinesin family has shown that kinesin-2, kinesin-9, and the more recently identified, kinesin-‘orphan’ III (also referred to as kinesin-16) and kinesin-17, are only found in organisms that are ciliated at some point during the life cycle [28, 36]. Members of these kinesin families can be found in ciliated plants such as Chlamydomonas, the moss Physcomitrella, and the water fern Marsilea, except kinesin-17, which is only present in Chlamydomonas [28, 36–38]. The genome of Arabidopsis, a flowering plant that is never makes ciliated cells, contains none of these kinesins [39]. Due to the reduction of cilia and IFT proteins in land plants, the majority of research on ciliogenesis in plants has been conducted in Chlamydomonas. It is somewhat ironic that the conserved 9?+?2 microtubule organization of motile axonemes was first observed in the spermatozoid of a fern [40].
Here, we are using spermatogenesis in Marsilea as a model for ciliogenesis. This provides us the unique ability to study the construction, organization and motility of ciliary apparatus produced in gametes of a land plant. Moreover, this gametohyte provides insights on the mechanisms that evolved to regulate de novo ciliogenesis in specialized cells of otherwise nonmotile organisms. Transcripts that encode members of the kinesin-2 and kinesin-9 families were selected as targets for our studies on the regulation of ciliogenesis in spermatids of Marsilea. mRNAs that encode kinesin-2 and kinesin-9 increase in abundance during the stage of development associated with spermatid shaping and ciliogenesis [38]. These late rises in transcript abundance led us to suspect that kinesin-2 and kinesin-9 may play critical roles for spermatid differentiation and ciliogenesis. Unlike many other systems, the male gametophyte of Marsilea has only one kinesin-2 that is apparent in its transcriptome. It is most similar to the kinesin-2 found in Physcomitrella and is divergent from the typical heterotrimeric kinesin-2 associated with IFT. In this study, we show that MvKinesin-2 is required for two separate events in this gametophyte. It is necessary for cytokinesis in spermatogenous cells and is also important for regulating the length of cilia during later phases of spermatid maturation. The Marsilea gametophyte has two transcripts that encode members of the kinesin-9 family; one is similar to the kinesin-9A and the other is most like kinesin-9B. In the gametophyte, we show that MvKinesin-9A is involved in the proper positioning of basal bodies that are required for ciliogenesis and it is necessary for motility. MvKinesin-9B is needed for the timely differentiation of motile spermatozoids in the rapidly developing gametophyte.