A dynamic DUO of regulatory proteins coordinates gamete specification and germ cell mitosis in the angiosperm male germline

The production of two functional sperm cells within each male gametophyte is essential for double fertilization in flowering plants and involves a single mitotic division of the male germ cell and cell specification to produce functional gametes. Several proteins that are important regulators of male germ cell division have been identified as well as the R2R3 MYB protein DUO1 that has a dual role in cell division and cell specification. We recently identified a novel regulatory protein DUO3, that has overlapping roles with DUO1 in cell division and specification and shows similarity to GON4 related cell lineage regulators in animals. DUO3 also has important roles outside the germline and is required for embryo patterning and meristem function. We outline the regulatory roles of DUO3 in male germline development and its possible mechanisms of action as a lineage regulator in current models that link germ cell cycle control and gamete specification.Key words: DUO3, male germline development, cell cycle, cell specification, Arabidopsis, pollen, GON4-LThe two sperm cells required for double fertilization in flowering plants are produced after an asymmetric division of the haploid microspore produces a large vegetative cell and a smaller germ cell, thereby establishing the male germline (reviewed in ref. 1; Fig. 1A). The germ cell is engulfed within the vegetative cell cytoplasm where it divides to produce the two sperm cells. The germ cell also goes through a process of specification, with ∼6,000 genes expressed in sperm cells,² many of which show specific or enhanced expression in the male germline and/or are essential for fertilization.^2–4 Since 2005 a number of proteins with important regulatory roles in either germ cell division^5–9 or both germ cell division and specification^10–12 have been described, enabling the formulation of basic models for the regulation of male germline development.^12,13 In our recent publication¹⁴ we identify a novel regulatory protein, DUO POLLEN3 (DUO3) that has essential roles in germ cell division and specification, as well as vital sporophytic functions. Here we present the role of DUO3 in an emerging model for the regulation of male germline development in Arabidopsis (Fig. 1B) and briefly discuss the wider role and possible mechanism of DUO3 function.Open in a separate windowFigure 1Overview of male gametophyte development in arabidopsis (a) and model of germ cell cycle progression and specification in the male germline (B). (A) Schematic of the distinct morphological stages of male gametophyte development in arabidopsis. Diploid pollen mother cells undergo meiotic division to produce a tetrad of haploid microspores. the released microspores undergo a highly asymmetric division to produce a bicellular pollen grain with a small germ cell engulfed within the cytoplasm of a large vegetative cell. Whilst the vegetative cell exits the cell cycle, the germ cell undergoes a further mitotic division to produce twin sperm cells. (B) a schematic model integrating the control of cell proliferation and sperm cell specification in male germline development of arabidopsis. after microspore division, the cell cycle inhibitors KrP6 and KrP7 are present in the newly formed germ cell. transient expression of FBL17 leads to the degradation of these KRPs, allowing CDKA/CYCD to phosphorylate RBR, thereby relieving RBR-mediated repression of the E2F/DP pathway and progression of the germ cell through S-phase. Gamete specification begins shortly after germ cell division, where the co-expression of DUO1 and DUO3 in the germ cell leads to the activation of common and distinct germline differentiation genes. Once S-phase is complete, the DuO1-dependent activation of the G₂/m phase regulator CYCB1;1 promotes germ cell cycle progression and entry into mitosis. In parallel, DUO3 also controls G₂/m transition, by an unknown mechanism that acts independently of cYcB1;1 expression. DUO1 and DUO3 therefore integrate germline differentiation with cell cycle progression. Ultimately, the cooperation of these parallel pathways results in a pair of fully differentiated sperm cells equipped with a complement of essential germline factors such as GcS1 that are required for successful gamete fusion.     

Polarity in Stem Cell Division: Asymmetric Stem Cell Division in Tissue Homeostasis

Many adult stem cells divide asymmetrically to balance self-renewal and differentiation, thereby maintaining tissue homeostasis. Asymmetric stem cell divisions depend on asymmetric cell architecture (i.e., cell polarity) within the cell and/or the cellular environment. In particular, as residents of the tissues they sustain, stem cells are inevitably placed in the context of the tissue architecture. Indeed, many stem cells are polarized within their microenvironment, or the stem cell niche, and their asymmetric division relies on their relationship with the microenvironment. Here, we review asymmetric stem cell divisions in the context of the stem cell niche with a focus on Drosophila germ line stem cells, where the nature of niche-dependent asymmetric stem cell division is well characterized.Asymmetric cell division allows stem cells to self-renew and produce another cell that undergoes differentiation, thus providing a simple method for tissue homeostasis. Stem cell self-renewal refers to the daughter(s) of stem cell division maintaining all stem cell characteristics, including proliferation capacity, maintenance of the undifferentiated state, and the capability to produce daughter cells that undergo differentiation. A failure to maintain the correct stem cell number has been speculated to lead to tumorigenesis/tissue hyperplasia via stem cell hyperproliferation or tissue degeneration/aging via a reduction in stem cell number or activity (Morrison and Kimble 2006; Rando 2006). This necessity changes during development. The stem cell pool requires expansion earlier in development, whereas maintenance is needed later to sustain tissue homeostasis.There are two major mechanisms to sustain a fixed number of adult stem cells: stem cell niche and asymmetric stem cell division, which are not mutually exclusive. Stem cell niche is a microenvironment in which stem cells reside, and provides essential signals required for stem cell identity (Fig. 1A). Physical limitation of niche “space” can therefore define stem cell number within a tissue. Within such a niche, many stem cells divide asymmetrically, giving rise to one stem cell and one differentiating cell, by placing one daughter inside and another outside of the niche, respectively (Fig. 1A). Nevertheless, some stem cells divide asymmetrically, apparently without the niche. For example, in Drosophila neuroblasts, cell-intrinsic fate determinants are polarized within a dividing cell, and subsequent partitioning of such fate determinants into daughter cells in an asymmetric manner results in asymmetric stem cell division (Fig. 1B) (see Fig. 3A and Prehoda 2009).Open in a separate windowFigure 1.Mechanisms of asymmetric stem cell division. (A) Asymmetric stem cell division by extrinsic fate determinants (i.e., the stem cell niche). The two daughters of stem cell division will be placed in distinct cellular environments either inside or outside the stem cell niche, leading to asymmetric fate choice. (B) Asymmetric stem cell division by intrinsic fate determinants. Fate determinants are polarized in the dividing stem cells, which are subsequently partitioned into two daughter cells unequally, thus making the division asymmetrical. Self-renewing (red line) and/or differentiation promoting (green line) factors may be involved.In this review, we focus primarily on asymmetric stem cell divisions in the Drosophila germ line as the most intensively studied examples of niche-dependent asymmetric stem cell division. We also discuss some examples of stem cell division outside Drosophila, where stem cells are known to divide asymmetrically or in a niche-dependent manner.     

An extracellular matrix protein prevents cytokinesis failure and aneuploidy in the C. elegans germline

Interactions between extracellular matrix (ECM) proteins and their transmembrane receptors mediate cytoskeletal reorganization and corresponding changes in cell shape during cell migration, adhesion, differentiation and polarization. Cytokinesis is the final step in cell division as cells employ a contractile ring composed of actin and myosin to partition one cell into two. Cells undergo dramatic changes in cell shape during the division process, creating new membrane and forming an extracellular invagination called the cleavage furrow. However, existing models of cytokinesis include no role for the ECM. In a recent paper, we demonstrate that depletion of a large secreted protein, hemicentin, results in membrane destabilization, cleavage furrow retraction and cytokinesis failure in C. elegans germ cells and in preimplantation mouse embryos.Here, we demonstrate that cytokinesis failure produces tetraploid intermediate cells with multipolar spindles, providing a potential explanation for the large number of aneuploid progeny observed among C. elegans hemicentin mutant hermaphrodites.Key words: aneuploidy, cytokinesis, extracellular matrix, C. elegans, cleavage furrow, hemicentin, tetraploid intermediateThe karyotype of C. elegans has five autosomes and one or two X chromosomes in males and hermaphrodites, respectively. The majority of self-progeny produced by wild-type hermaphrodites are hermaphrodites (∼99.8%), while rare meiotic nondisjunction of the X chromosome produces nullo-X gametes and 0.2% males. Mutations in over 30 genes result in a 10–150-fold increase in the frequency of males among hermaphrodite self-progeny, due to increases in defects in X chromosome segregation.¹ The majority of these ‘him’ (high incidence of males) loci are genes that encode proteins associated with the intracellular machinery of meiotic chromosome segregation.^2,3 Unique among him genes, the him-4 locus encodes hemicentin, a large, highly conserved component of the extracellular matrix (ECM).⁴ In addition to defects in germline chromosome segregation, him-4 mutants have pleiotropic defects in somatic cell adhesion and migration.^1,4 The extracellular distribution of hemicentin at cell junctions that are defective in him-4 mutants dovetails with current models of cell adhesion and migration.⁵ However, it leaves unexplained several questions about how a secreted ECM component promotes correct chromosome segregation in the C. elegans germline.C. elegans hermaphrodite gonads are composed of two U-shaped tubes, and gametogenesis proceeds sequentially from the distal to the proximal end of each tube. Germ cells in C. elegans have incomplete cleavage furrows that connect them to a central cytoplasmic core, allowing distal cells to act as “nurses” while allowing more mature proximal oocytes to fill with bulk cytoplasm.^6–8 Several genetic and cytogenetic observations suggest a mitotic rather than a meiotic origin for germline chromosome segregation defects observed in the absence of hemicentin.⁴ For example, jackpots of male progeny from individual hermaphrodites and nullisomy of primary meiocytes in him-4 mutants suggest a defect in a mitotic germline stem cell rather than in a post-mitotic process. Our recent work describing hemicentin localization at the cleavage furrows of dividing cells in the early mouse embryo and C. elegans germline, in addition to membrane destabilization, cleavage furrow retraction and cytokinesis failure in the absence of hemicentin, suggests that hemicentin has an evolutionarily conserved role in stabilizing and preventing retraction of nascent cleavage furrows.⁹Aneuploid cells are frequently observed in, and may be associated with the generation of, human tumor cells. Recent work from several laboratories suggests that cytokinesis failure is one of several mechanisms whereby tumor cells generate tetraploid intermediates that result in the production of aneuploid daughter cells in subsequent cell divisions. One proposed mechanism for the generation of aneuploid daughter cells from a tetraploid intermediate is thought to involve multipolar mitotic spindles that result in asymmetric mitoses.^10–13To determine whether a similar mechanism might be responsible for the aneuploidy observed in the absence of hemicentin, him-4 (rh319) animals were examined for multipolar mitotic spindles. A significant fraction (14%) of mitotic germ cells have multipolar spindles that are not observed in a wild-type background (Fig. 1 and Fig. 1).Open in a separate windowFigure 1Multinucleate germ cells and multipolar germ cells observed in the mitotic zone of him-4 mutant hermaphrodites. (A) PH::RFP and histone::GFP in the mitotic region of wild-type (left) and him-4 (rh319) hermaphrodite gonads. Large numbers of multinucleate cells are observed among mitotic germ cells in mutant gonads (arrows). (B) PH::RFP and tubulin::GFP in the mitotic region of wild-type (left) and him-4 (rh319) hermaphrodite gonads. A significant fraction (14^–16.

Table 1

Severity and types of defective germ cells in him-4 gonads

The Histone Deacetylase Inhibitor Trichostatin A Promotes Totipotency in the Male Gametophyte

The haploid male gametophyte, the pollen grain, is a terminally differentiated structure whose function ends at fertilization. Plant breeding and propagation widely use haploid embryo production from in vitro–cultured male gametophytes, but this technique remains poorly understood at the mechanistic level. Here, we show that histone deacetylases (HDACs) regulate the switch to haploid embryogenesis. Blocking HDAC activity with trichostatin A (TSA) in cultured male gametophytes of Brassica napus leads to a large increase in the proportion of cells that switch from pollen to embryogenic growth. Embryogenic growth is enhanced by, but not dependent on, the high-temperature stress that is normally used to induce haploid embryogenesis in B. napus. The male gametophyte of Arabidopsis thaliana, which is recalcitrant to haploid embryo development in culture, also forms embryogenic cell clusters after TSA treatment. Genetic analysis suggests that the HDAC protein HDA17 plays a role in this process. TSA treatment of male gametophytes is associated with the hyperacetylation of histones H3 and H4. We propose that the totipotency of the male gametophyte is kept in check by an HDAC-dependent mechanism and that the stress treatments used to induce haploid embryo development in culture impinge on this HDAC-dependent pathway.     

Cytokinetic astralogy

Division plane specification in animal cells has long been presumed to involve direct contact between microtubules of the anaphase mitotic spindle and the cell cortex. In this issue, von Dassow et al. (von Dassow et al. 2009. J. Cell. Biol. doi:10.1083/jcb.200907090) challenge this assumption by showing that spindle microtubules can effectively position the division plane at a distance from the cell cortex.Cell division, or cytokinesis, is accomplished via constriction of an equatorially localized contractile ring composed of filamentous actin and myosin II (Rappaport, 1996). Accurate division plane specification is essential to properly partition the cytoplasm and permit each daughter cell to receive a single copy of the genome. To ensure this accuracy, microtubules of the mitotic spindle signal to the cell cortex upon anaphase onset and promote assembly of the contractile ring between the separating chromosomes. The precise mechanism by which microtubules position the contractile ring, however, remains elusive.Early models on the nature of the spindle-derived signal proposed that astral rays (later found to be microtubules) position the division plane by either locally promoting contractility at the cell equator or inhibiting contractility at the cell poles (Rappaport, 1996). Recent evidence, though, suggests that distinct microtubule populations within a single cell provide multiple signals to promote accurate division (Canman et al., 2003; Bringmann and Hyman, 2005; Chen et al., 2008; Foe and von Dassow, 2008; von Dassow, 2009).The anaphase mitotic spindle contains several subtypes of microtubules, each of which is likely to contribute to division plane specification. Although kinetochore microtubules drive chromosome segregation during anaphase, nonkinetochore microtubules extend and maintain close proximity with the assembling central spindle (Mastronarde et al., 1993). Central spindle microtubules are highly stable (Salmon et al., 1976) and organize into an antiparallel bundled array between the separating chromosomes (Mastronarde et al., 1993). Preventing central spindle assembly usually results in a complete failure in cytokinesis, and prevents division plane specification in many cell types (Glotzer, 2005). Astral microtubules, however, are highly dynamic and grow out circumferentially from the centrosomes toward the cell cortex. Increasing evidence suggests that the astral microtubule signal inhibits contractility (see below; Canman et al., 2000; Kurz et al., 2002; Lewellyn et al., 2009).Regardless of the mechanism of division plane specification via microtubules, nearly all current models depend on direct contact between microtubules of the mitotic spindle and the cell cortex. Most of these models were based on observations that at the time of division plane specification, astral microtubules contact the cell cortex in nearly all systems studied. Nonkinetochore and/or central spindle microtubules have also been proposed to deliver critical contractile signals to the cell equator (Murata-Hori and Wang, 2002; Canman et al., 2003; Somers and Saint, 2003; Verbrugghe and White, 2004; Lewellyn et al., 2009; Vale et al., 2009). Yet in many cell types (especially early embryos), central spindle microtubules are at some distance from the cell cortex during division plane specification. Despite this, signal delivery for both astral and central spindle microtubules was proposed to occur via direct transport along microtubules to the cell cortex. The study of von Dassow et al. in this issue, however, indicates that accurate division plane specification does not require any close microtubule/cortical contact and may occur via a diffusion-based mechanism (see also Salmon and Wolniak, 1990).By treating echinoderm and Xenopus embryos with controlled levels of trichostatin A (TSA), which destabilizes acetylated dynamic microtubules via inhibition of the tubulin deacetylase HDAC6 (Matsuyama et al., 2002), von Dassow et al. (2009) were able to preferentially prevent astral microtubule growth while leaving central spindle microtubules intact. TSA treatment did not block anaphase onset or central spindle assembly, but resulted in the complete disruption of all direct microtubule contact with the cell cortex. Nevertheless, TSA-treated cells were able to undergo cytokinesis successfully (Fig. 1 A). The lack of astral microtubules in TSA-treated cells was also recapitulated by double centrosome ablation, and again the cells were able to undergo cytokinesis (Fig. 1 B). In both experiments, cytokinesis occurred in a timely manner, but the contractile ring was broader than in control cells. Together, these data suggest that spindle microtubules are sufficient to provide a diffusible stimulatory signal capable of defining the cell division plane without any direct contact with the cell cortex (von Dassow et al., 2009).Open in a separate windowFigure 1.Testing models of division plane specification by targeting distinct microtubule populations. By selectively eliminating astral microtubules with either controlled TSA-treatment (A) or by double centrosome ablation (B), von Dassow et al. (2009) provide strong evidence that microtubule contact with the cell cortex is not essential for successful cytokinesis. When a single centrosome was ablated, the division plane was displaced away from the ablated aster (B); this suggests that astral microtubules provide an inhibitory signal. Further, anucleate cells would only complete cytokinesis if the intracentrosomal distance exceeded the distance from the centrosomes to the cell cortex (C).The authors noticed that cytokinesis occurred selectively at a position with reduced microtubule density in control cells; therefore, they explored the role of astral microtubules in division plane positioning. By selectively ablating one centrosome just before anaphase onset, von Dassow et al. (2009) were also able to provide strong support for an inhibitory role of astral microtubules in division plane specification. When a single centrosome was ablated, the division plane was displaced away from the remaining astral microtubules and toward the ablated centrosome (Fig. 1 B). Further evidence for an inhibitory role of astral microtubules in cytokinesis came from close examination of the intracentrosomal distance in anucleate cells that were able to undergo cytokinesis relative to those that were not. Cells were only able to undergo cytokinesis when the intracentrosomal distance exceeded the distance from the centrosomes to the cell cortex (Fig. 1 C), which suggests that cytokinesis requires an aster-free zone. The authors propose a mechanism in these anucleate cells whereby global activation of contractility drives division plane specification refined by a zone of astral separation (von Dassow et al., 2009). However, one possibility is that a central spindle still forms in these anucleate cells and thus provides the same diffusion-based signal that promotes division in cells without asters. Indeed, antiparallel arrays of bundled microtubules that resemble the central spindle are known to form between asters without intervening chromosomes in other systems (Savoian et al., 1999).To summarize, the results described by von Dassow et al. (2009) support a model in which central spindle microtubules provide a diffusible stimulatory signal to promote the assembly of a broad contractile ring, which is then refined by astral microtubules into a tight contractile ring. It is tempting to speculate on the molecular nature of this diffusible signal and mechanism of the astral refinement during cytokinesis. Signaling via the small GTPase Rho is required for cytokinesis and is dependent on spindle microtubules (Bement et al., 2005; Piekny et al., 2005). von Dassow et al. (2009) showed that in TSA-treated cells lacking astral microtubules, the equatorial zone of active Rho GTPase is broader relative to control cells. Rho activation is promoted (at least in part) via the central spindle–localized GTP exchange factor, ECT2 (Glotzer, 2005). In parallel, the GTPase-activating protein (GAP) CYK4/MgcRacGAP also associates with the central spindle, where it acts to both limit the zone of Rho activity (Miller and Bement, 2009) and to promote the inactivation of another small GTPase, Rac (D''Avino et al., 2004; Yoshizaki et al., 2004; Canman et al., 2008). Perhaps in parallel to central spindle mediated activation of Rho signaling, local inactivation of the inhibitory Rac signal via CYK4 GAP activity would further specify the division plane, even at a distance (Fig. 2). When the dynamic asters are present, they could then additionally amplify Rac signaling at the cell poles via a similar mechanism to what occurs during cell motility (Wittmann and Waterman-Storer, 2001). This local feedback loop would reinforce the positive signal coming from the central spindle via Rho activation and could help delimit active Rho at the cell equator (Fig. 2). Certainly, understanding how Rho activation can be propagated to the cell cortex via diffusion in such an accurate manner will be a major future challenge.Open in a separate windowFigure 2.Model for central spindle–mediated signaling via Rho family small GTPases. Central spindle–localized guanine nucleotide exchange factor ECT2 leads to Rho activation at the cell equator. At the same time, central spindle–localized CYK4 (a Rho family GAP) would also locally inactivate the inhibitory Rac signal. Further refinement of the zone of active Rho by astral microtubule–activated Rac could then sharpen the Rho zone into a tight contractile ring.     

Deciphering the Physalis floridana Double-Layered-Lantern1 Mutant Provides Insights into Functional Divergence of the GLOBOSA Duplicates within the Solanaceae

Glycosylphosphatidylinositol-anchor biosynthesis and glycosylphosphatidylinositol modification of proteins are central to coordinated plant development.Since their discovery (Low and Saltiel, 1988), glycosylphosphatidylinositol-anchored proteins (GPI-APs) have provoked intense interest as crucial regulators for growth, morphogenesis, reproduction, and disease pathogenesis in organisms ranging from yeast and trypanosomes to animals and plants. The lipid moiety, the glycosylphosphatidylinositol (GPI) anchor, is synthesized in the endoplasmic reticulum (ER); the protein component is cotranslationally inserted into the ER and posttranslationally modified by the addition of a GPI anchor (Kinoshita et al., 2013; Fig. 1). GPI-APs are then transported via the Golgi to the outer surface of the plasma membrane. The lipid anchor mediates stable attachment of these proteins to the cell surface, where some play important roles as signaling regulators from sphingolipid- and sterol-enriched membrane microdomains (Simons and Gerl, 2010). Some GPI-APs are released from the cell membrane by phosphatidylinositol-specific phospholipases to the extracellular matrix, where they might engage in processes such as cell adhesion and cell-cell communication. In Arabidopsis (Arabidopsis thaliana), there are about 250 predicted GPI-APs (Borner et al., 2003), a relatively large number compared with about 150 in mammals and 50 in the budding yeast (Saccharomyces cerevisiae). Important functions for plant GPI-APs have been elucidated through the study of individual proteins, such as the COBRA family in cell expansion and cell wall biosynthesis (Brady et al., 2007), ARABINOGALACTAN PROTEIN18 in megagametogenesis (Demesa-Arévalo and Vielle-Calzada, 2013), and LORELEI in the pollen tube-female gametophyte interaction (Capron et al., 2008; Tsukamoto et al., 2010; Duan et al., 2014). However, it is the studies of mutants defective in GPI biosynthesis that underscore the general importance of GPI-APs as a class: lacking the capacity to assemble the anchor is lethal.Open in a separate windowFigure 1.GPI anchor biosynthesis pathway. Ten or 11 stepwise modifications of phosphoinositide occur, starting from the synthesis of N-glucosamine-phosphoinositide on the cytoplasmic surface of the ER, followed by its flipping to the ER lumenal side for additional modifications, ending with the addition of the terminal ethanolamine phosphate. Proteins destined for GPI modification are synthesized with a C-terminal signature sequence recognized by the GPI transamidase (a five-protein-enzyme complex) that concomitantly cleaves the peptide at what is designated as the ω and ω+1 amino acids and attaches the GPI anchor in a transamination reaction (red arrows). The GPI-modified proteins are then sorted and transported via the Golgi apparatus to the cell membrane. The established biosynthetic proteins from Arabidopsis and their mammalian homologs are indicated; the galactosylation step appears to be plant specific. The diagram is modeled after figure 3 in Ellis et al. (2010), which also provided a complete list of potential plant orthologs of the human and yeast proteins in the pathway.The GPI anchor is synthesized by an elaborate biosynthetic pathway, starting on the cytoplasmic side of the ER and ending with a completely assembled core anchor on the lumenal surface of the ER (Fig. 1). Prior to their transport out of the ER, proteins destined for GPI modification are cleaved at a C-terminal signature sequence by a GPI transamidase complex that in two enzymatic steps concomitantly attaches a GPI anchor to the C terminus of processed proteins (Kinoshita, 2014). Most of the knowledge on GPI biosynthesis and GPI-AP modification is derived from studies in mammals and yeast, but the pathway is likely to be conserved in plants (Ellis et al., 2010). In a recent article in Plant Physiology, Dai et al. (2014) reported that a GPI anchor biosynthesis mutant, abnormal pollen tube guidance1 (atpg1), displays both embryo lethality and severely depressed male fertility. They determined that APTG1 is homologous to the yeast GPI10 and human PIG-B (for phosphatidylinositol glycan anchor biosynthesis) proteins, the last of three distinct mannosyltransferases that modify the precursor anchor (Fig. 1), and showed that APTG1 can functionally substitute for GPI10 in a conditionally lethal gpi10 yeast mutant. Previous studies have demonstrated that Arabidopsis SETH1 (a male fertility god in Egyptian mythology), SETH2, and PEANUT1 (PNT1), encoding homologs of mammalian PIG-C, PIG-A, and PIG-M (Fig. 1) and their corresponding yeast counterparts, respectively, are important for male fertility (Lalanne et al., 2004; Gillmor et al., 2005). In addition, loss of the first mannosyltransferase in the pathway in pnt1 results in early seedling lethality. pnt1 embryos are severely defective, displaying various cell division anomalies and exhibiting altered levels and ectopic deposition of cell wall polymers. The results reported by Dai et al. (2014), therefore, further demonstrate the conservation of the GPI biosynthesis pathway and the importance of GPI anchoring in plant development and reproduction.The aptg1 mutant was isolated in a search for mutants defective in pollen tube targeting of ovules (Fig. 2), an intriguing and crucial step in plant reproduction. A pollen tube is guided to an ovule by attractants, and upon reaching the target, the female gametophyte, the pollen tube ruptures, ejecting its cytoplasm and releasing sperm for fertilization (Dresselhaus and Franklin-Tong, 2013). aptg1 pollen tubes either fail to target ovules or undertake a more twisted pathway before entering an ovule. In an earlier study, Li et al. (2013) showed that a GPI-AP, COBRA-LIKE10 (COBL10), is required to maintain normal pollen tube growth rates and ovule targeting efficiency. In aptg1 pollen tubes, citrine fluorescent protein-COBL10 was absent from its normal apical membrane location while the citrine fluorescent signal in the cytoplasm was more intense, implying that the diminished presence of COBL10 on the apical membrane could be an underlying cause for the ovule-targeting phenotype. This observation also demonstrates that GPI anchoring is important for the subsequent sorting and transport of these proteins to their destined locations (Kinoshita et al., 2013) and consistent with a wholesale failure of GPI-APs to reach their functional locations as underlying lethality in GPI biosynthesis mutants.Open in a separate windowFigure 2.Pollen tube targeting of ovules in an Arabidopsis pistil. GUS-expressing pollen grains pollinated the pistil. Each blue dot represents discharged cytoplasm from a pollen tube that, in response to attractants, has successfully targeted the ovule and penetrated the female gametophyte and was induced to burst. The cytoplasmic discharge releases sperm for fertilization.While it is clear that major biological roles are played by GPI-APs, many questions remain. Most constituents of the plant GPI anchor biosynthetic pathway remain to be functionally established (Fig. 1). Much has been said about the membrane environments where GPI-APs are localized, but we are far from understanding the precise roles they play in assembling these domains and regulating their functional dynamics. Advances in high-resolution imaging at the cell surface and biochemical approaches to determine the constituents in these membrane microdomains (Simons and Gerl, 2010) should accelerate our understanding of the importance of GPI anchoring as a conserved strategy among eukaryotes to control a wide range of processes.     

Orientation of vascular cell divisions in Arabidopsis

Orientation of cell division is essential for plant development as the direction of growth is determined by the direction of cell expansion and orientation of cell division. We have demonstrated that cell division orientation in vascular tissue is regulated by the interactions between a receptor kinase (PXY) expressed in dividing cells and its peptide ligand (CLE41) that is localized to adjacent phloem cells. Given that other receptor kinases have been identified as orienting the cell division plane in several developmental processes, we suggest that localized signaling from adjacent cells may be a general mechanism for defining the plane of cell division.Key words: xylem, phloem, cell division orientation, procambium, cambiumThroughout the life of plants, new organs are generated from meristems which contain stem cells at their center. Meristematic cells divide in regulated processes resulting in displacement of daughter cells to the periphery of the meristem where they differentiate, taking on new cell identities.¹ Vascular meristems (cambium and procambium) are responsible for radial growth and are the main source of plant biomass.² Their regulation has a come under increasing scrutiny as biomass is likely to play an increasing role in generation of renewable energy.³Arabidopsis vascular tissue is organized into discrete collateral bundles in stems,⁴ whereas in hypocotyls, vasculature forms in a continuous ring, much like that of trees.⁵ In both cases spatially separated xylem and phloem are formed along the stem mediolateral axis and are populated with cells derived from the procambium or cambium (Fig. 1). Vascular initials displaced from the meristematic zone towards the center of the stem differentiate into xylem whereas those displaced towards the outside of the stem differentiate into phloem. This organization occurs because vascular meristematic cells are long and thin and divide periclinally down their long axis, perpendicular to the mediolateral axis. Because these are highly ordered divisions, vascular tissue is characterized by long files of cells. Until recently regulatory factors which influence the highly ordered nature of these divisions—and therefore plant vascular tissue organization were entirely unknown.Open in a separate windowFigure 1Arabidopsis vascular tissue at the base of inflorescence stems (A) and hypocotyls (B). The mediolateral axes are marked with arrows, x is xylem, ph is phloem, pc is procambium, c is cambium. Scale bars are 50 µm.     

AUXIN RESPONSE FACTOR17 Is Essential for Pollen Wall Pattern Formation in Arabidopsis

In angiosperms, pollen wall pattern formation is determined by primexine deposition on the microspores. Here, we show that AUXIN RESPONSE FACTOR17 (ARF17) is essential for primexine formation and pollen development in Arabidopsis (Arabidopsis thaliana). The arf17 mutant exhibited a male-sterile phenotype with normal vegetative growth. ARF17 was expressed in microsporocytes and microgametophytes from meiosis to the bicellular microspore stage. Transmission electron microscopy analysis showed that primexine was absent in the arf17 mutant, which leads to pollen wall-patterning defects and pollen degradation. Callose deposition was also significantly reduced in the arf17 mutant, and the expression of CALLOSE SYNTHASE5 (CalS5), the major gene for callose biosynthesis, was approximately 10% that of the wild type. Chromatin immunoprecipitation and electrophoretic mobility shift assays showed that ARF17 can directly bind to the CalS5 promoter. As indicated by the expression of DR5-driven green fluorescent protein, which is an synthetic auxin response reporter, auxin signaling appeared to be specifically impaired in arf17 anthers. Taken together, our results suggest that ARF17 is essential for pollen wall patterning in Arabidopsis by modulating primexine formation at least partially through direct regulation of CalS5 gene expression.In angiosperms, the pollen wall is the most complex plant cell wall. It consists of the inner wall, the intine, and the outer wall, the exine. The exine is further divided into sexine and nexine layers. The sculptured sexine includes three major parts: baculum, tectum, and tryphine (Heslop-Harrison, 1971; Piffanelli et al., 1998; Ariizumi and Toriyama, 2011; Fig. 1A). Production of a functional pollen wall requires the precise spatial and temporal cooperation of gametophytic and sporophytic tissues and metabolic events (Blackmore et al., 2007). The intine layer is controlled gametophytically, while the exine is regulated sporophytically. The sporophytic tapetum cells provide material for pollen wall formation, while primexine determines pollen wall patterning (Heslop-Harrison, 1968).Open in a separate windowFigure 1.Schematic representation of the pollen wall and primexine development. A, The innermost layer adjacent to the plasma membrane is the intine. The bacula (Ba), tectum (Te), and tryphine (T) make up the sexine layer. The nexine is located between the intine and the sexine layers. The exine includes the nexine and sexine layers. B, Primexine (Pr) appears between callose (Cl) and plasma membrane (Pm) at the early tetrad stage (left panel). Subsequently, the plasma membrane becomes undulated (middle panel) and sporopollenin deposits on the peak of the undulated plasma membrane to form bacula and tectum (right panel).After meiosis, four microspores were encased in callose to form a tetrad. Subsequently, the primexine develops between the callose layer and the microspore membrane (Fig. 1B), and the microspore plasma membrane becomes undulated (Fig. 1B; Fitzgerald and Knox, 1995; Southworth and Jernstedt, 1995). Sporopollenin precursors then accumulate on the peak of the undulated microspore membrane to form the bacula and tectum (Fig. 1B; Fitzgerald and Knox, 1995). After callose degradation, individual microspores are released from the tetrad, and the bacula and tectum continue to grow into exine with further sporopollenin deposition (Fitzgerald and Knox, 1995; Blackmore et al., 2007).The callose has been reported to affect primexine deposition and pollen wall pattern formation. The peripheral callose layer, secreted by the microsporocyte, acts as the mold for primexine (Waterkeyn and Bienfait, 1970; Heslop-Harrison, 1971). CALLOSE SYNTHASE5 (CalS5) is the major enzyme responsible for the biosynthesis of the callose peripheral of the tetrad (Dong et al., 2005; Nishikawa et al., 2005). Mutation of Cals5 and abnormal CalS5 pre-mRNA splicing resulted in defective peripheral callose deposition and primexine formation (Dong et al., 2005; Nishikawa et al., 2005; Huang et al., 2013). Besides CalS5, four membrane-associated proteins have also been reported to be involved in primexine formation: DEFECTIVE EXINE FORMATION1 (DEX1; Paxson-Sowders et al., 1997, 2001), NO EXINE FORMATION1 (NEF1; Ariizumi et al., 2004), RUPTURED POLLEN GRAIN1 (RPG1; Guan et al., 2008; Sun et al., 2013), and NO PRIMEXINE AND PLASMA MEMBRANE UNDULATION (NPU; Chang et al., 2012). Mutation of DEX1 results in delayed primexine formation (Paxson-Sowders et al., 2001). The primexine in nef1 is coarse compared with the wild type (Ariizumi et al., 2004). The loss-of-function rpg1 shows reduced primexine deposition (Guan et al., 2008; Sun et al., 2013), while the npu mutant does not deposit any primexine (Chang et al., 2012). Recently, it was reported that Arabidopsis (Arabidopsis thaliana) CYCLIN-DEPENDENT KINASE G1 (CDKG1) associates with the spliceosome to regulate the CalS5 pre-mRNA splicing for pollen wall formation (Huang et al., 2013). Clearly, disrupted primexine deposition leads to aberrant pollen wall patterning and ruptured pollen grains in these mutants.The plant hormone auxin has multiple roles in plant reproductive development (Aloni et al., 2006; Sundberg and Østergaard, 2009). Knocking out the two auxin biosynthesis genes, YUC2 and YUC6, caused an essentially sterile phenotype in Arabidopsis (Cheng et al., 2006). Auxin transport is essential for anther development; defects in auxin flow in anther filaments resulted in abnormal pollen mitosis and pollen development (Feng et al., 2006). Ding et al. (2012) showed that the endoplasmic reticulum-localized auxin transporter PIN8 regulates auxin homeostasis and male gametophyte development in Arabidopsis. Evidence for the localization, biosynthesis, and transport of auxin indicates that auxin regulates anther dehiscence, pollen maturation, and filament elongation during late anther development (Cecchetti et al., 2004, 2008). The role of auxin in pollen wall development has not been reported.The auxin signaling pathway requires the auxin response factor (ARF) family proteins (Quint and Gray, 2006; Guilfoyle and Hagen, 2007; Mockaitis and Estelle, 2008; Vanneste and Friml, 2009). ARF proteins can either activate or repress the expression of target genes by directly binding to auxin response elements (AuxRE; TGTCTC/GAGACA) in the promoters (Ulmasov et al., 1999; Tiwari et al., 2003). The Arabidopsis ARF family contains 23 members. A subgroup in the ARF family, ARF10, ARF16, and ARF17, are targets of miRNA160 (Okushima et al., 2005b; Wang et al., 2005). Plants expressing miR160-resistant ARF17 exhibited pleiotropic developmental defects, including abnormal stamen structure and reduced fertility (Mallory et al., 2005). This indicates a potential role for ARF17 in plant fertility, although the detailed function remains unknown. In addition, ARF17 was also proposed to negatively regulate adventitious root formation (Sorin et al., 2005; Gutierrez et al., 2009), although an ARF17 knockout mutant was not reported and its phenotype is unknown.In this work, we isolated and characterized a loss-of-function mutant of ARF17. Results from cytological observations suggest that ARF17 controls callose biosynthesis and primexine deposition. Consistent with this, the ARF17 protein is highly abundant in microsporocytes and tetrads. Furthermore, we demonstrate that the ARF17 protein is able to bind the promoter region of CalS5. Our results suggest that ARF17 regulates pollen wall pattern formation in Arabidopsis.     

Symmetry Breaking in Plants: Molecular Mechanisms Regulating Asymmetric Cell Divisions in Arabidopsis

Asymmetric cell division generates cell types with different specialized functions or fates. This type of division is critical to the overall cellular organization and development of many multicellular organisms. In plants, regulated asymmetric cell divisions are of particular importance because cell migration does not occur. The influence of extrinsic cues on asymmetric cell division in plants is well documented. Recently, candidate intrinsic factors have been identified and links between intrinsic and extrinsic components are beginning to be elucidated. A novel mechanism in breaking symmetry was revealed that involves the movement of typically intrinsic factors between plant cells. As we learn more about the regulation of asymmetric cell divisions in plants, we can begin to reflect on the similarities and differences between the strategies used by plants and animals. Focusing on the underlying molecular mechanisms, this article describes three selected cases of symmetry-breaking events in the model plant Arabidopsis thaliana. These examples occur in early embryogenesis, stomatal development, and ground tissue formation in the root.Plant cells are surrounded by rigid cell walls that prevent cell movement. Precisely oriented cell division is especially important in the absence of cell migration. When a plant cell divides, a new wall forms between the daughter cells; this permanently delineates their relative positions. As a consequence, the selection of the division plane is crucial for plant tissue organization and overall organ architecture.The selection of the division plane is unique in plants compared with other organisms (for a detailed review of plant cytokinesis see Jürgens 2005). In budding yeast, landmarks at previous bud-sites position the division plane (see Slaughter et al. 2009), whereas in fission yeast it is positioned by the nucleus. The position of the centrosomes and the mitotic spindle determine division plane orientation in animal cells (Balasubramanian et al. 2004; see Munro and Bowerman 2009). In plants, the division plane is positioned by a ring of microtubules and F-actin, called the preprophase band that forms at the cell periphery (Mineyuki 1999). Despite these striking differences, similarities are beginning to emerge. For example, in budding yeast and animals, GTPase activation plays an important role in positioning division planes (Balasubramanian et al. 2004; see McCaffrey and Macara 2009; Slaughter et al. 2009; Munro and Bowerman 2009; Prehoda 2009). In plants, a GTPase was recently found to be localized at the preprophase band and may provide spatial information during division (Xu et al. 2008). Therefore, common molecular mechanisms may underlie division plane selection in plants and animals.In all organisms, asymmetric cell division is a common method to obtain distinct cell types from a single progenitor cell. Before an asymmetric division a cell must first establish an internal asymmetry; this process is termed breaking symmetry. Breaking symmetry can be generally defined as the asymmetric organization of cellular components along one axis leading to cellular polarization as a prerequisite to division. An asymmetric cell division can generate two cells of different morphology (i.e., size and/or shape) or different specialized functions or fates. Specifically, the products of asymmetric cell divisions fall into two major categories. In one case, a mother cell can divide asymmetrically to produce a daughter cell to replace it and a daughter cell with a distinct fate, as in the case of stem cell divisions. Alternatively, a mother cell can divide asymmetrically to produce two distinct daughter cells at the expense of mother cell identity. Following division, there are two basic mechanisms by which a mother cell generates daughters that are distinct from itself. One mechanism is intrinsic to the cell and is defined by segregation of cellular determinants before division. The other mechanism is extrinsic to the cell and is defined by external cues that direct daughter cell fate (Horvitz and Herskowitz 1992). Intrinsic and extrinsic mechanisms are not necessarily independent and often work in combination to direct asymmetric division and specification of daughter cells.The first zygotic division of fucoid brown algae, such as Fucus, is one of the earliest studied examples of symmetry breaking in plants regulated by both extrinsic and intrinsic mechanisms. Fucus zygotes are an excellent organism in which to examine the cellular biology underlying asymmetric cell division (for a recent review see Homble and Leonetti 2007). In the presence of extrinsic cues, such as light, the asymmetric zygotic division is oriented perpendicular to the light gradient so that one cell resides on the “shady” side of the gradient (Kropf 1992, 1997; Alessa and Kropf 1999). In the absence of environmental cues, the division is oriented relative to the sperm entry site and therefore appears random within a field of cells (Hable and Kropf 2000). This indicates that Fucus zygotes have an intrinsic mechanism to break symmetry in the absence of extrinsic cues. This intrinsic mechanism occurs rapidly after fertilization, however it can later be overridden by extrinsic cues. Although much is known about the role of the cytoskeleton, membrane depolarization, and ion flux in Fucus embryo polarization, the molecular components regulating these events remain unknown because of a lack of molecular tools. To address the molecular mechanisms regulating asymmetric cell divisions in plants, the use of a more genetically tractable plant is necessary.In the model plant Arabidopsis thaliana, many genes that are involved in asymmetric cell division have been identified. Interestingly, many of these genes are thought to function in both asymmetric division and subsequent cell fate specification suggesting that, in plants, these processes are tightly linked. Despite these advances, relatively little is known about the cellular processes of breaking symmetry in plants. Because of these two caveats, the term breaking symmetry must be defined more generally in plants. We use this term to describe a process in which a cell divides asymmetrically to form a daughter cell with a distinct fate. Here we discuss three examples in which the molecular mechanisms that participate in symmetry-breaking events in plants are beginning to be elucidated. These examples occur in early embryogenesis, formation of stomata, and root development (Fig. 1). We confine the topics of this article to Arabidopsis thaliana because the majority of the experimental work, particularly using molecular genetics, is focused on this species.Open in a separate windowFigure 1.An Arabidopsis plant. Schematic of an adult plant depicting roots, leaves, stems, and flowers. In this article, three symmetry-breaking events are discussed. These examples are taken from different organs of the plant. The first example is taken from embryo development, which occurs after fertilization of the flower in the silique (seed pod) in Arabidopsis. Next, symmetry breaking in the leaf epidermis during stomatal development is discussed. Finally, the asymmetric cell division that gives rise to the ground tissue in the root is considered.     

The force and effect of cell proliferation

EMBO J 32: 2790–2803 doi:10.1038/emboj.2013.197; published online September102013The spatiotemporal control of cell divisions is a key factor in epithelial morphogenesis and patterning. Mao et al (2013) now describe how differential rates of proliferation within the Drosophila wing disc epithelium give rise to anisotropic tissue tension in peripheral/proximal regions of the disc. Such global tissue tension anisotropy in turn determines the orientation of cell divisions by controlling epithelial cell elongation.Oriented cell divisions play important roles in the establishment of the animal body plan by both influencing tissue morphogenesis and generating cellular diversity. Generally, the direction of the cell division plane is determined by the orientation of the mitotic spindle prior to cytokinesis. The observation that the mitotic spindle in most animal cell types aligns with the cell''s longest axis has led to the formulation of the ‘long-axis-rule'', postulating that cell shape anisotropy is the main determinant of spindle orientation (Minc et al, 2011). However, cell shape anisotropy is unlikely to be the only determinant since many cell types round up during mitosis, thereby losing their shape anisotropy and others do not follow the long-axis-rule at all. In such cases, division orientation is determined by the polarizing activity of biochemical signals originating from the environment (reviewed in Morin and Bellaïche, 2011). In addition, externally applied forces have also been suggested to control division orientation of single cells in culture independently from their effect on cell shape (Fink et al, 2011).Epithelial growth implies that cells divide parallel to the tissue plane with both daughter cells remaining integrated within the tissue. Although it has been recognized that defects in apico-basal polarity lead to spindle misalignment and disruption of epithelial architecture, the molecular mechanisms underlying this regulation are still unknown. Recent work in the Drosophila wing disc epithelium uncovered that the junctional proteins Scribbled and Discs large 1 (Dlg1) are required for proper spindle alignment parallel to the tissue plane (Nakajima et al, 2013). Similarly, in the Drosophila follicular epithelium, spindle orientation is dependent on the lateral localization of Dlg1, independently of its role in apico-basal polarity (Bergstralh et al, 2013). While such mechanisms ensure that cells divide parallel to the epithelial plane, other mechanisms must still be present to determine the orientation of the mitotic spindle within this plane.In the Drosophila wing disc epithelium, symmetric cell divisions preferentially align with the proximal-distal (PD) axis, thus elongating the organ along this axis (Baena-López et al, 2005). This preferential cell division orientation is determined by the Fat-Dachsous pathway, which promotes accumulation of the atypical myosin Dachs at PD cellular junctions. The polarized activity of Dachs in turn drives cell elongation along the PD axis, leading to a preferential orientation of the mitotic spindle along this axis (Mao et al, 2011). In this issue of The EMBO Journal, Mao et al (2013) report that while mitotic cells located in central regions of the wing disc indeed elongate and divide along the PD axis, cells located in the periphery (proximal edge) elongate and divide orthogonally to the PD axis (Figure 1). These results suggested some type of global planar tissue polarization in proximal regions of the wing disc overriding the local effects of Dachs on spindle orientation. By using laser ablation to reveal tissue tension, the authors showed that in peripheral/proximal regions of the wing disc, junctions oriented orthogonal to the PD axis (PD junctions) are under higher tension than junctions oriented along this axis (lateral junctions; Figure 1). This led them to hypothesize that anisotropic tissue tension might control division orientation of proximal wing cells. Through a combination of elegant genetic experiments and theoretical modelling, the authors then demonstrated that this global tension anisotropy in the proximal wing disc arises from higher cell division rates observed in central versus proximal regions of the wing disc. Furthermore, this apparent tension anisotropy causes concentric elongation of proximal wing disc cells orienting their mitotic spindle orthogonal to the PD axis (Figure 1).Open in a separate windowFigure 1Differential rates of cell division between distal (green) and proximal (red) regions of the Drosophila wing disc epithelium (1) give rise to global patterns of tension anisotropy within the tissue (2). This tension anisotropy promotes cell elongation along the main axis of tension, thereby controlling the orientation of cell division via cell shape anisotropies in proximal regions of the wing disc (3); D, distal; P, proximal.Collectively, these results demonstrate that differential proliferation rates within a tissue can generate global tension anisotropies, which promote cell shape changes that again influence cell division orientation. Further dissection of the mechanisms by which tissue tension controls cell division orientation will clarify if anisotropic tension controls division orientation solely through cell elongation, or if additional mechanosensing mechanisms exist that more directly convey tissue tension information to the mitotic spindle. It might also be worth exploring whether cell divisions along the main axis of tension within the wing disc affect global tension anisotropy, and whether the formation of anisotropic tension around areas of cell proliferation affects the rate of cell division therein. Such interplay between tissue tension anisotropy and cell division orientation/rate will likely be critical for maintaining physiological degrees of tissue tension and growth.In general, the work by Mao et al (2013) provides compelling evidence for a functional link between tissue tension and cell division orientation in a physiological relevant context, paving the way for future studies addressing the reciprocal relationship between these two aspects in tissue morphogenesis.     

discs large in the Drosophila testis: An old player on a new task

Gamete development requires a coordinated soma-germ line interaction that ensures renewal and differentiation of germline and somatic stem cells. The physical contact between the germline and somatic cell populations is crucial because it allows the exchange of diffusible signals among them. The tumor suppressor gene discs large (dlg) encodes a septate junction protein with functions in epithelial cell polarity, asymmetric neuroblast division and formation of neuromuscular junctions. Our recent work reveals a new role of dlg in the Drosophila testis, as mutations in dlg lead to testis defects and cell death. Dlg is required throughout spermatogenesis in the somatic lineage and its localization changes from a uniform distribution along the plasma membrane of somatic cells in the testis apex, to a restricted localization on the distally located somatic cell in growing cysts. The extensive defects in dlg testis underline the importance of the somatic cells in the establishment and maintenance of the male stem cell niche and somatic cell differentiation. Here, we discuss our latest findings on the role of dlg in the Drosophila testis, supporting the view that junction proteins are dynamic structures, which can provide guiding cues to recruit scaffold proteins or other signaling molecules.Key words: dlg, Drosophila melanogaster, germ cell differentiation, septate junctions, somatic stem cells, somatic cyst cellsThe discovery of mutations causing neoplasia during Drosophila development¹ followed by the molecular characterization of these genes has shown that cell polarity is critically affected in the tumor cells. Three of these genes, lethal (2) giant larvae (lgl), discs large-1 (dlg) and scribble (scrib), encode scaffolding proteins, associated with either the cytoskeleton matrix or septate junctions.^2–9 Analysis over the last decades revealed that these proteins act as more than just static barriers limiting the diffusion of other components along the cortical cell domains. In particular, they function as dynamic organizing centers, targeting site-specific proteins in discrete domains and provide guiding cues for signaling molecules and insertion of membrane components.¹⁰ Nowadays, several studies place Dlg as a key player in numerous tissues at different time points throughout development, contributing to epithelial polarity establishment, polarized membrane insertion, asymmetric neuroblast division, formation of neuromuscular junctions (NMJ) and planar cell polarity in vertebrates.^{4,7,9,11–15} Interestingly, the four mammalian homologs of the Drosophila dlg are also involved in cell polarity and become downregulated in a series of human cancers. Moreover, a mammalian dlg-1 transgene can substitute a defective dlg gene in Drosophila and rescue the development of dlg mutant animals.^8,9,16 Therefore, it is perfectly plausible to envisage that Drosophila functions uncovered in other tissues may be similarly conserved in other species.Similar to vertebrates, the Drosophila testis consists of germ cells and somatic cells. The somatic cells of the hub form the organizing center at the apex of the testis and recruit germline stem cells (GSCs) (Fig. 1A), giving rise to the male stem cell niche. Each GSC attached to the hub is surrounded by two somatic stem cells (SSCs). Upon asymmetric stem cell division, each GSC produces a new GSC attached to the hub and a distally located gonialblast, whereas each SSC pair divides to generate two SSCs and two somatic cyst cells (SCCs).^17–19 The gonialblast divides mitotically four more times to give rise to 16 interconnected spermatogonial cells, forming a cyst surrounded by the two SCCs.²¹ Then, the spermatogonial cyst grows markedly in size and differentiates to primary spermatocytes that enter the pre-meiotic phase (Fig. 1A, B and D–F).¹⁹ We have recently investigated a new role of dlg in the Drosophila testis.²⁰ In contrast to the overgrowth phenotypes observed in imaginal discs and brain hemispheres,^4,6,21 dlg inactivation leads to testis degeneration during early larval development. The dlg testes are extremely small and contain a reduced number of GSCs loosely attached to the hub (Fig. 1C).²⁰ In addition, the few spermatogonial cysts, which become formed, fully degenerate during the second and third larval instars.Open in a separate windowFigure 1(A) Diagram depicting early spermatogenesis in Drosophila. The red line indicates the Dlg distribution in the hub, SSCs, early and late SCCs. GSCs, germline stem cells; SCCs, somatic cyst cells; SSCs, somatic stem cells. (B) Apex of wild-type 3^rd instar larval testis and (C) dlg^m52 3^rd instar larval testis displaying a reduced number of GSCs, spermatogonial and spermatocyte cysts. In mutant dlg^m52 testes, SSCs and early SCCS positively stained for Traffic-jam are still present. However, late SCCs identified by staining for Eye absent remain undetectable.²⁰ Vasa (red), Traffic-jam (green) and Arm + α-Spectrin (blue). (D–F) Pattern of Dlg distribution in 3^rd instar larval testis. (E and F) are enlargements at different optical sections of the testis shown in (D), displaying Dlg staining in the hub region and growing spermatocyte cysts, respectively. Testis hub is oriented towards the left.Recent advances in Drosophila spermatogenesis and the male stem cel niche have clearly shown that the intrinsic signals of the germ cells are important but not sufficient to support stem cell homeostasis. Signals emanating from SSCs and SCCs are also required for testis development. Physical contacts among the cell populations in the Drosophila testis allow the exchange of signals, which promote tissue survival and set the balance between stem cell identity and differentiation.¹⁸ Interestingly, the Dlg protein is present in all somatic cells including the hub, SSCs and SCCs (Fig. 1D–F) and a specific requirement of dlg in these cells is further supported by the finding that the mutant phenotype could be reverted by expressing dlg in somatic cells but not in germ cells.²⁰ Further analysis points out that the mutant GSCs are significantly larger than in wild-type, lower in number and loosely attached to the hub.²⁰ Preliminary results indicate a defective orientation of the daughter centrosome and absence of mitotic spindle in dividing GSCs, which together with the increased GSC size, allows us to speculate that GSCs may grow but fail to undergo mitosis. Similar phenotypes are observed in mutations affecting the insulin pathway,²² further stressing the importance of cell communication between germ cells and somatic cells. However, a functional connection between dlg and the insulin pathway remains yet to be experimentally determined. The defects detected in the dlg mutant testis place dlg as a key regulator in the early development of spermatogonial cysts.During testis differentiation, the Dlg protein displays a dynamic change in its intracellular localization. First, Dlg is uniformly associated with the plasma membrane on all somatic cells in the male stem cell niche and early spermatogonial cysts, and then becomes restricted to the most proximal SCC in late spermatogonial cysts and growing spermatocyte cysts (Fig. 1D–F). The transition from a uniform to a restricted distribution is achieved between the 8- to 16-cell cyst stages, when one of the two SCCs caps the distal side of the growing cyst. Interestingly, the capping corresponds to the axis of cyst growth and points out the direction of cyst expansion. A restoration of nearly normal testis morphology can be obtained by expressing a dlg transgene in SSCs and early SCCs. In contrast, expression of a dlg transgene in later SCCs can still restore the development of already formed cysts, some of which may reach an advanced post-meiotic stage, but the testis is generally depleted in early cysts.²⁰ These data indicate that dlg is required for the differentiation of the somatic cell lineage and, therefore, the early differentiation of the germline into spermatogonial cells. Results of RNAi experiments provide also evidence that dlg silencing in late SSCs results in a fragmentation of the cysts in advanced stages.²⁰ The specific recruitment of Dlg on the membrane of distal SCCs remains an open question, although it is possible to envisage that phosphorylation of Dlg by the PAR-1 kinase may play a role, as it has been shown in the case of postsynaptic targeting of Dlg in NMJs.²³Therefore, Dlg may exert different functions in the somatic cells that are required for (1) GSC attachment to the hub and proper asymmetric GSC division, (2) the architecture and early differentiation of the spermatogonial cysts and (3) the expansion and growth of the spermatocyte cysts. Presumably, dlg is required for establishing and maintaining a tight connection between GSCs and SSCs around the hub. The connection between gonialblast and SCC is also maintained during the mitotic divisions. In SSCs and early SCCs, dlg acts critically to establish a normal cyst structure, whereas in further spermatogonial and spermatocyte stages dlg is critical for the survival, growth and expansion of the cyst. Our rescue experiments further suggest that if proper cyst architecture is not established when the two stem cell populations move away from the hub, it cannot be re-established at later stages. Moreover, the restricted Dlg localization in the distal SCC suggests that dlg may be necessary for the polarized growth of spermatocyte cysts and thus act as a critical factor for planar cell polarity. In the second phase, dlg is involved in spermatogonial and spermatocyte cyst growth, viability and differentiation. Further RNA silencing experiments using GAL4-drivers that target dlg in SCCs during late spermatocyte growth, meiosis and post-meiotic stages may further provide insights into dlg requirement during the whole spermatogenesis. Preliminary results indicate that Dlg is similarly produced and localized on the distal SCC in spermatocyte and spermatid cysts of adult testes, suggesting that dlg may be required from the early stages, from the establishment of male stem cell niche and SCC survival, up to the later stages of sperm formation.An unexpected finding of our analysis deals with the formation of wavy and ruffled plasma membrane in dlg overexpressing cells capping the spermatocyte cysts. One way to interpret this result would be to consider that Dlg regulates the intensity of germ cell encapsulation through the Egfr pathway, which is the major signaling pathway active at the microenvironment of the spermatogonial cysts.^24,25 Membrane ruffling, detected in somatic cells upon dlg overexpression, is highly reminiscent of the formation of lammellipodia-like structures formed upon upregulation of Rac1 in SCCs.²⁶ Rac1 is a downstream component of the Egfr pathway and acts antagonistically to Rho to regulate germ cell encapsulation. As the Dlg protein plays a central role in the organization of epithelial junctions and in signal transduction at sites of cell-cell contact, it is possible to envisage that the C-terminal tail of Egfr interacts with one of the PDZ domains of Dlg.⁹ In this way, dlg inactivation would result in a disruption of the Egfr protein complexes, block the Egfr pathway and impair Rac1 function. Based on these data, we hypothesize that Dlg may act on the cytoskeleton of the somatic cells to mediate cell-shape changes leading to either cellular extensions over the spermatogonial and spermatocyte cysts or reinforcing cell-to-cell contact with the growing germ cells.A second possibility would imply a general role for Dlg in membrane proliferation and expansion of the SCCs. It has already been shown that Dlg regulates membrane proliferation in a subset of NMJ in a dose-dependent fashion.²⁷ Recent focus on membrane growth during cellularization indicates again that Dlg is an important player in the process of polarized membrane insertion.^11,28–30 Up to now, there is no mechanism describing how SCCs in Drosophila testis expand, elongate and envelop germ cell cysts, and how the SCCs direct sperm differentiation and individualization. Membrane proliferation during tissue spreading and cell surface extensions is frequently associated with the formation of membrane ruffles.^31,32 The finding that dlg overexpression in the distal SCC leads to membrane ruffling indicates that Dlg may mediate membrane growth and membrane extension over the cysts but not necessarily at the expense of the proximal SCC devoid of Dlg. Therefore, there should be a physical limitation in the expansion of the dlg-expressing cell, independent of the amount of synthesized Dlg. Further analyses of components at the junctions between the distal and proximal SCCs or components exhibiting a complementary distribution to Dlg may provide ways to identify further regulators of testis morphogenesis.If Dlg defines sites of membrane addition it may provide a link between membrane trafficking and insertion of polarized membrane components. In NMJs, the postsynaptic distribution of the t-SNARE protein Gtaxin depends on its direct interaction to the Dlg GUK domain,¹² whereas in early embryogenesis Dlg genetically interacts with Exo84.³³ Moreover, the Dlg-Strabismus complex recruits membrane associated proteins and lipids from internal membranes to sites of new plasma membrane formation.¹¹ The occurrence of similar proteins in testis was reported in humans where the SNARE-associated component Snapin binds Pumilio2 and Nanos1 proteins in the male germ cells.³⁴ It would be interesting to know whether Dlg plays a similar role in Drosophila testis, in guiding t-SNARE proteins and components of the exocyst complex into intracellular membranes, either directly or indirectly by regulating the distribution of their direct binding partners. Although Dlg may bind to different proteins in epithelial cells, neuroblasts and NMJ according to the protein availability in these tissues, the function of the Dlg protein may be still conserved in a broader sense. Through its PDZ domains Dlg may bind to numerous transmembrane proteins and receptors, and may link them to the cytoskeleton or signaling pathways. The knowledge gained on the role of Dlg in these systems will allow us to study how Dlg mediates membrane proliferation in the early germ cells in male gonads.Recent work has showed that Zero population growth (Zpg), the Drosophila gap junction Innexin 4, is localized to the spermatogonia surface, primarily on the sides adjacent to SCCs³⁵ and is required for the survival and differentiation of early germ cells in both sexes.^35–37 In zpg testes, the spermatogonia are unable to differentiate and are progressively lost, leading to the formation of tiny testes containing a small number of GSCs and germline clusters devoid of branching fusome,³⁵ resembling the dlg phenotype. In contrast, the SCCs that die through apoptosis in dlg testes are present in zpg, indicating that Dlg acts primarily on SCCs and Zpg on the germ cells.^20,35 Moreover, zpg testes display often a considerably enlarged hub. However, a direct comparison of the effect of the two proteins on the hub cannot be made because the null dlg^m52 allele produces a truncated non-functional Dlg protein that could still be detected in the hub.²⁰ Apparently this protein, which contains the PDZ1 and PDZ2 domains, could be recognized by a monoclonal antibody against the PDZ2 domain (data not shown).²⁰ This observation raises the possibility that the truncated Dlg protein may maintain some of its binding properties, which prevents the hub structure from falling apart. Further studies will be performed to determine the requirement of dlg in hub formation and structure.Our results, complementary to current researches conducted in this field, point out the importance of the somatic cell contribution in the organization of the Drosophila testis and the differentiation of the male germline. In mammals, spermatogenesis depends also on interactions between somatic Sertoli cells and germ cells. Sertoli cells act as supportive somatic cells and contain junction proteins with a high degree of similarity to Dlg. These proteins play a critical role in mammalian spermatogenesis.^38,39 Furthermore, the identification of mammalian genes with known function in Drosophila spermatogenesis and the evolutionary conservation among the Dlg proteins suggests that the pathways regulating the balance between stem cell renewal and differentiation might be similarly conserved. Interestingly, recent observations in mammals indicate that Dlg homologs play a role in the formation of mouse gonads and interact with gap junction proteins.^13,40 In addition, Dlg is required for smooth muscle orientation in the mouse ureter¹³ and interacts with the gap protein Connexin 32,⁴¹ whereas ZO-1, a MAGUK protein bearing similarity to Dlg and associated with tight junctions in mammalian Sertoli cells,³⁹ binds also to gap junction proteins, among them connexin 43, which is the predominant gap junction protein in the testis.^38,39,42 All these observations point out to functional similarities between Drosophila and vertebrate Dlg and provide strong indications that our findings in Drosophila may be extended to higher organisms.