The Interactive Fly
Genes involved in tissue and organ development
Development of the male germline stem cell niche in Drosophila
Asymmetric inheritance of mother versus daughter centrosome in stem cell division
A role for actin dynamics in individualization during spermatogenesis
Cytoskeletal dynamics in male meiosis
The search for Y-linked genes: Y chromosome fertility factors encode dynein heavy chain polypeptides
Stem cells are found in specialized microenvironments, or 'niches', which regulate stem cell identity and behavior. The adult testis and ovary in Drosophila contain germline stem cells (GSCs) with well-defined niches, and are excellent models for studying niche development. This study investigates the formation of the testis GSC niche, or 'hub', during the late stages of embryogenesis. By morphological and molecular criteria, the development of an embryonic hub that forms from a subset of anterior somatic gonadal precursors (SGPs) were identified and followed in the male gonad. Embryonic hub cells form a discrete cluster apart from other SGPs, express several molecular markers in common with the adult hub and organize anterior-most germ cells in a rosette pattern characteristic of GSCs in the adult. The sex determination genes transformer and doublesex ensure that hub formation occurs only in males. Interestingly, hub formation occurs in both XX and XY gonads mutant for doublesex, indicating that doublesex is required to repress hub formation in females. This work establishes the Drosophila male GSC niche as a model for understanding the mechanisms controlling niche formation and initial stem cell recruitment, as well as the development of sexual dimorphism in the gonad (Le Bras, 2006).
The evidence indicates that an embryonic hub, which appears to give rise to the adult hub and create the male GSC niche, forms during the late stages of embryogenesis. A subset of anterior SGPs initiates expression of several molecular markers that are also expressed in the adult hub. These SGPs segregate into a tight cluster in a distinct region of the gonad, and a subset of germ cells organizes around these SGPs in a manner similar to the organization of GSCs around the adult hub. Since spermatogenesis begins by early larval stages, it is possible that the embryonic hub already forms a functional GSC niche. The formation of the hub, or indeed any stem cell niche, can be divided into the distinct issues of niche cell identity, niche morphogenesis, and stem cell recruitment (Le Bras, 2006).
The data indicate that the specification of hub cell identity occurs in two stages. During the first stage, some SGPs acquire an anterior identity that is sexually dimorphic, as indicated by the male-specific expression of esg and upd. Anterior SGP identity is positively regulated by abd-A, and is repressed by Abd-B, while sexual identity is regulated by tra and dsx. During the second stage of hub cell specification, a subset of these anterior SGPs acquires hub cell identity during stage 17 of embryogenesis. Only some anterior SGPs maintain esg expression, and the control of late gene expression in the hub appears to be distinct from early expression in anterior SGPs, since some esg and upd enhancer traps only exhibit gonad expression in the hub at this later stage. Furthermore, cells that maintain esg expression during stage 17 also express every other marker of adult hub identity tested, including Fasciclin 3, cdi, DN-cadherin and DE-cadherin. It is concluded that these cells are specified as hub cells at this time. The fate of the anterior SGPs that lose esg expression and do not form part of the hub is unknown. An intriguing possibility is that these cells could form another important somatic cell type: the cyst progenitor cells (somatic stem cells) that associate with the hub along with the GSCs (Le Bras, 2006).
Based on its expression pattern, the transcription factor esg would seem to be an excellent candidate for specifying hub cell identity. However, no changes were observed in the expression of other hub markers in esg null mutants; this includes expression of DE-cadherin, which is known to be regulated by esg in other tissues. It has been reported, however, that esg is required for hub maintenance, and that the hub is severely defective at later stages in esg mutants that survive embryogenesis. Thus, esg is critical for the male GSC niche, but is either not important for the initial formation of this structure, or acts redundantly with another factor (Le Bras, 2006).
It has been possible to follow the morphogenesis of the hub from the time of gonad formation until the embryonic hub is fully formed. At the time of gonad coalescence, anterior SGPs interact with other SGPs, and with the germ cells, in a manner that is indistinguishable from posterior SGPs. However, during stage 17, the hub cells undergo dramatic changes in their relationship to other SGPs and germ cells. Hub cells segregate away from other SGPs to one pole of the gonad, and coalesce tightly with one another. In addition, hub cells do not ensheath the germ cells at this stage. Instead, a defined interface between hub cells and germ cells forms which is labeled by DE- and DN-cadherin, but not Fasciclin 3. Thus, hub cells appear to maximize their interactions with one another, and minimize their interactions with other cells in the gonad, although they clearly still contact a subset of germ cells (Le Bras, 2006).
It is apparent that the changes in cell–cell contact and morphology that occur during hub formation require changes in cell adhesion. Indeed, characteristic changes have been found in expression of the homophilic adhesion molecules Fasciclin 3, DN-cadherin and DE-cadherin occur during hub formation; all three are significantly upregulated in the embryonic and adult hub. Increased homophilic adhesion among hub cells could account for their ability to maximize their contacts with one another, and sort away from other SGPs. However, no changes were observed in embryonic hub formation in mutants for these cell adhesion molecules. Thus, these proteins, and possibly others, may act redundantly in this process (Le Bras, 2006).
It is clear that a subset of germ cells organizes specifically with the developing hub as it forms. During the last stage of hub formation, germ cells become oriented in a rosette distribution around the developing hub in a manner characteristic of GSCs in the adult. These may represent the subset of germ cells that will become GSCs. The presence of DE- and DN-cadherin at sites of hub–germ cell contact suggests that cadherin-mediated adhesion may be important for niche–GSC interaction in the testis, as has been observed in the ovary. Interestingly, germ cells are not required for hub formation. Analysis of a number of hub identity markers indicates that these cell form normally from a subset of anterior SGPs in embryos that lack germ cells. The hub does not appear as well compacted in these embryos, consistent with observations of the adult hub, indicating that hub–germ cell contact (or hub–germ cell signaling) affects the final shape of the hub. Nevertheless, the GSC niche can form in the absence of one of its stem cell populations (somatic stem cells may still be present). It will be of great interest in the future to determine if the subset of germ cells organized around the male embryonic hub are, indeed, developing GSCs, and to study how their transition to stem cell identity might be regulated by the niche (Le Bras, 2006).
The formation of the male GSC niche is a sex-specific characteristic of anterior SGPs. Male-specific expression of esg and hub formation both require the sex determination genes tra and dsx. In some tissues, DSXM is required to promote male development and repress female development, while the opposite is true for DSXF. Interestingly, it was found that embryonic hub development is entirely masculinized in dsx null mutants; XX and XY individuals appear identical when mutant for dsx and both resemble wild type males. Thus, no role is seen for DSXM in promoting embryonic hub formation, while DSXF is required in females to repress hub formation. Since esg is expressed male-specifically, it is one candidate for being directly regulated by DSX (Le Bras, 2006).
We can compare the development of the anterior SGPs and hub with the development of another sexually dimorphic cell type, the msSGPs that join the posterior of the male gonad. First of all, these two cell types are distinct and do not depend on one another for their proper development. The hub still forms in Abd-B mutants that lack msSGPs, while msSGPs are still found in the gonad in Pc mutants, in which no anterior SGPs or hub cells form. Second, the mechanism for how sexual dimorphism is created differs between the two cell types. msSGPs are present only in males because they have undergone sex-specific apoptosis in females. In contrast, no apoptosis was observed in anterior SGPs. These cells appear to remain present in both sexes, but only form a hub in males. Thus, although the sex determination genes tra and dsx regulate sex-specific development of both cell types, the cellular mechanisms employed are different. Finally, as was observed for the hub, development of the msSGPs is completely masculinized in dsx mutant embryos. Thus, for both of these cell types, the male pattern of development in the embryonic gonad is the default state in the absence of dsx function, and it is the role of DSXF to repress male development in females. However, DSXM may well play a role in development of one or both of these gonad cell types at later stages, since proper testis development in males clearly requires dsx (Le Bras, 2006).
The sex determination pathway must also ensure that GSC niches form in females and are different from those in males. Recently, it has been shown that germ cells populating the anterior of the gonad in female embryos are predisposed to become GSCs in the adult ovary, while germ cells populating the posterior rarely become GSCs. This suggests that anterior SGPs in the female embryonic gonad may promote GSC identity, similar to what is proposed to happen in the male during hub formation. One possibility is that anterior SGPs give rise to GSC niches in both sexes, while genes such as tra and dsx control whether these niches will be male or female (Le Bras, 2006).
In conclusion, the development has been followed of the embryonic hub, which may represent the nascent GSC niche for the testis. This work provides a basis for further understanding the mechanisms controlling niche formation and GSC recruitment in Drosophila, and determining if these mechanisms are conserved in other stem cell systems, including the GSC niche of the mammalian testis (Le Bras, 2006).
Adult stem cells often divide asymmetrically to produce one self-renewed stem cell and one differentiating cell, thus maintaining both populations. The asymmetric outcome of stem cell divisions can be specified by an oriented spindle and local self-renewal signals from the stem cell niche. This study shows developmentally programmed asymmetric behavior and inheritance of mother and daughter centrosomes underlies the stereotyped spindle orientation and asymmetric outcome of stem cell divisions in the Drosophila male germ line. The mother centrosome remains anchored near the niche while the daughter centrosome migrates to the opposite side of the cell before spindle formation (Yamashita, 2007).
Adult stem cells maintain populations of highly differentiated but short-lived cells throughout the life of the organism. To maintain the critical balance between stem cell and differentiating cell populations, stem cells have a potential to divide asymmetrically, producing one stem and one differentiating cell. The asymmetric outcome of stem cell divisions can be specified by regulated spindle orientation, such that the two daughter cells are placed in different microenvironments that either specify stem cell identity (stem cell niche) or allow differentiation. Drosophila male germline stem cells (GSCs) are maintained through attachment to somatic hub cells, which constitute the stem cell niche. Hub cells secrete the signaling ligand Upd, which activates the Janus kinase-signal transducer and activator of transcription (JAK-STAT) pathway in the neighboring germ cells to specify stem cell identity. Drosophila male GSCs normally divide asymmetrically, producing one stem cell, which remains attached to the hub, and one gonialblast, which initiates differentiation. This stereotyped asymmetric outcome is controlled by the orientation of the mitotic spindle in GSCs: The spindle lies perpendicular to the hub so that one daughter cell inherits the attachment to the hub, whereas the other is displaced away (Yamashita, 2007).
The stereotyped orientation of the mitotic spindle is set up by the positioning of centrosomes during interphase. GSCs remain oriented toward the niche throughout the cell cycle. In G1 phase, the single centrosome is located near the interface with the hub. When the duplicated centrosomes separate in G2 phase, one stays next to the hub, whereas the other migrates to the opposite side of the cell. Centrosomes in the GSCs separate unusually early in interphase, rather than at the G2-prophase transition, so it is common to see GSCs with fully separated centrosomes without a spindle (Yamashita, 2007).
Differences between the mother and daughter centrosomes underlie the stereotyped behavior of the centrosomes in Drosophila male GSCs. The mother centrosome normally remains anchored to the hub-GSC interface and is inherited by the GSC, whereas the daughter centrosome moves away from the hub and is inherited by the cell that commits to differentiation. Mother and daughter centrosomes were differentially labeled by transient expression of green fluorescent protein-pericentrin/AKAP450 C-terminus (GFP-PACT) from the Drosophila pericentrin-like protein under heat shock-Gal4 control. The PACT domain, which is necessary and sufficient for centriolar localization, is incorporated into centrioles only during centrosome duplication and does not exchange with the cytoplasmic pool. Both the mother and daughter centrosomes are labeled by GFP-PACT in the first cell cycle after heat shock. In the second cell cycle, the daughter centrosome retains GFP-PACT, whereas the mother centrosome is not labeled, thus distinguishing the mother and daughter centrosomes. After a short burst of GFP-PACT expression induced by a 2.5-hour heat shock, 20% - 30% of the GSCs had GFP-labeled centrosomes, indicating the duplication of centrosomes during the window of GFP-PACT expression. By 12 hours after heat shock, >90% of the labeled GSCs had two GFP-positive centrosomes, indicating that they had progressed to the G2 phase of the first cell cycle after GFP-PACT incorporation (Yamashita, 2007).
By 18 to 24 hours after heat shock, the number of GSCs with two GFP-positive centrosomes had decreased, whereas the number of GSCs with one GFP-positive and one GFP-negative centrosome had increased, suggesting progression into the second cell cycle. Generally, the centrosome distal to the hub was labeled, whereas the centrosome proximal to the hub was GFP-negative, indicating that the daughter centrosomes migrate away from the hub-GSC interface during asymmetric GSC divisions (Yamashita, 2007).
Labeling the mother rather than the daughter centrosomes confirmed that the male GSCs in the niche preferentially retain mother centrosomes over time. Centrioles assembled during early embryogenesis were labeled using the NGT40 Gal4 driver to drive the expression of GFP-PACT in blastoderm-stage embryos, shutting off after germband extension. In the first cell cycle after the depletion of the cytoplasmic pool of GFP-PACT in the GSCs, both the mother and daughter centrosomes should be labeled. In subsequent cell cycles, only the mother centrosomes should be labeled (Yamashita, 2007).
In most GSCs in the second or later cell cycle after the depletion of cytoplasmic GFP-PACT, the labeled centrosome was positioned next to the hub-GSC interface, and the unlabeled centrosome had moved away from the hub. The frequency of GSCs that had the proximal, but not distal, centrosome labeled remained constant over time for 10 days (L3 larvae to day-3 adults), suggesting that the mother centrosomes are reliably retained by the GSCs, even through multiple rounds of GSC divisions. Some GSCs maintained cytoplasmic GFP-PACT, especially in L3 larvae, suggesting that the GFP-PACT had not yet been diluted out. Some GSCs with two labeled centrosomes were observed, suggesting that they are in the first cell cycle after the depletion of cytoplasmic GFP-PACT (Yamashita, 2007).
The mother centrosomes in GSCs appeared to maintain robust interphase microtubule arrays. Ultrastructural analysis of the GSCs revealed that the centrosome proximal to the hub was commonly associated with many microtubules throughout the cell cycle. Nineteen centrosomes in GSCs were scored in serial sections of the apical tips of five wild-type testes. Eleven centrosomes were localized close to the adherens junctions between the hub and the GSCs. Nine of these proximal centrosomes appeared to be in interphase cells, based on nuclear morphology and microtubule arrangement. Typically, these interphase centrosomes proximal to the hub were associated with numerous microtubules. In some samples, microtubules appeared to extend from the centrosome toward the adherens junctions. The other two proximal centrosomes appeared to be in cells in mitotic prophase, based on their robust microtubule arrays containing bundled microtubules running parallel to or piercing the nuclear surface (Yamashita, 2007).
In contrast, of the five distal centrosomes in the apparently interphase cells that were scored, four had few associated microtubules. The remaining three distal centrosomes appeared to be in cells in mitotic prophase, based on microtubule arrays containing bundled microtubules. Thus, the mother centrosomes may maintain interphase microtubule arrays that anchor them to the hub-GSC interface, whereas the daughter centrosomes may initially have few associated microtubules and be free to move, establishing a robust microtubule array only later in the cell cycle (Yamashita, 2007).
Consistent with the idea that astral microtubules anchor the mother centrosomes to the hub-GSC interface, mother- versus daughter centrosome positioning was randomized in GSCs that were homozygous mutant for centrosomin (cnn), an integral centrosomal protein required to anchor astral microtubules to centrosomes. Analysis of mother and daughter centrosomes after transient expression of GFP-PACT revealed that, for cnn homozygous mutant GSCs where one of the two centrosomes was positioned next to the hub, it was essentially random whether the mother or the daughter centrosome stayed next to the hub. In addition, in >25% of total labeled GSCs, neither of the two centrosomes was next to the hub (Yamashita, 2007).
These results indicate that the two centrosomes in Drosophila male GSCs have different characters and fates. The mother centrosome stays next to the junction with the niche and is inherited by the cell that self-renews stem cell fate. Thus, GSCs can maintain an old centriole assembled many cell generations earlier. In contrast, the daughter centrosome migrates away from the niche and is inherited by the cell that will initiate differentiation. It is postulated that the mother centrosomes in male GSCs may remain anchored to the GSC-niche interface throughout the cell cycle by attachment to astral microtubules connected to the adherens junction, whereas the daughter centrosomes may initially have few associated microtubules and thus can move away from the niche. Microtubule-dependent differential segregation of mother and daughter spindle-pole bodies (equivalent to centrosomes in higher organisms) is observed in budding yeast. In cultured vertebrate cells, the centrioles mature slowly over the cell cycle, and the mother centrosomes (containing a mature centriole) attach astral microtubules more effectively and are more stationary than daughter centrosomes in interphase. The unusually early separation of centrosomes in interphase male GSCs may provide a way to move the daughter centrosome out of range of the stabilizing influence of the adherens junction complex before it becomes competent to hold a robust microtubule array (Yamashita, 2007).
Developmentally programmed anchoring of the mother centrosome may provide a key mechanism to ensure the stereotyped orientation of the mitotic spindle and thus the reliably asymmetric outcome of the male GSC divisions. Although it is tempting to speculate that determinants associated with the mother or daughter centrosome may play a role in specifying stem cell or differentiating-cell fates, such determinants are yet to be identified. Rather, the asymmetric inheritance of mother and daughter centrosomes in male GSCs may be a consequence of the cytoskeletal mechanisms that are imposed as part of the stem cell program to anchor one centrosome next to the niche throughout the interphase, ensuring a properly oriented spindle (Yamashita, 2007).
During late stages of spermatogenesis in Drosophila, a cyst of 64 syncytial spermatids elongates as the sperm axonemes are formed inside it. Then this elongated cyst is remodeled into individual sperm by a process called individualization. At the start of individualization actin cones assemble around the spermatid nuclei and then synchronously move from the heads to the tips of the tails. As the actin cones move, a large accumulation of cytoplasm and vesicles, called the cystic bulge, forms around them. In the cystic bulge, the membrane of the cyst is remodeled to enclose each sperm axoneme. Individualization is especially interesting as a cell biological process because it requires an unusual amount of membrane remodeling using a well-defined actin structure. The fully elongated cyst can be up to 1800 microm long; therefore, this process requires the actin structures important for the process to move unidirectionally over a significant length. During the process, the bulk of the cytoplasm is discarded from the cell body. However, there is little information about the mechanism of this process (Noguchi, 2003).
In order to better understand the mechanism of sperm individualization, an in vitro culture system was developed in which live observation of individualization can be performed in isolated cysts. The whole process of individualization, during which a bundle of 64 syncytial spermatids is separated into individual sperm, takes place in these cultures. The speed of cystic bulge movement is fairly constant along the length of the cyst. Actin drugs, but not microtubule drugs inhibit cystic bulge movement, suggesting that the movement requires proper actin dynamics but not microtubules. GFP-tagged actin was expressed in the cyst and fluorescence recovery after photobleaching was monitored using confocal microscopy to analyze actin dynamics in cones. Actin turns over throughout the cone, with that at the leading edge of the cones turning over with slightly faster kinetics. Actin does not treadmill from the front to the back of the cone. Actin in moving actin cones turns over in about 12 minutes, although prior to onset of movement, turnover is much slower. Visualization of membrane using the dye FM1-43 reveals that the cystic bulge has an extremely complicated series of membrane invaginations and the transition from syncytial to individualized spermatids occurs at the front of the actin cones. It is also suggested that endocytosis and exocytosis might not be important for membrane remodeling. This system should be suitable for analysis of defects in male sterile mutants and for investigating other steps of spermatogenesis (Noguchi, 2003).
These data are most consistent with the idea that actin cone movement is driven by actin polymerization, similar to lamellipodia extension and Listeria motility. The speed of the cystic bulge movement is similar to the speed of movement of the leading edge of lamellipodia. Cystic bulge movement is altered very quickly after inhibiting either assembly or disassembly of actin, consistent with the requirement for active actin assembly and disassembly for movement. In fluorescence recovery after photobleaching (FRAP) experiments, a slightly faster rate of turnover at the front was detected than in the rear of the cone. It is likely that the faster dynamics of actin at the front is important for movement. In addition, the observed acceleration of actin dynamics after the onset of movement supports this idea. Arp 2/3 complex, which is the key factor involved in promoting actin polymerization at the leading edge, is enriched at the front of actin cones, suggesting that this site is important for force generation. All these data support the idea that the driving force is actin polymerization (Noguchi, 2003).
However, some puzzling differences in actin behavior in this structure when compared with leading edge protrusion make it difficult to explain how assembly drives movement in this case. (1) It had been expected that actin would treadmill through the actin cone from front to back, because of assembly at the front, i.e. in the direction of movement. This has been observed in other actin motility processes. However, this is not the case in actin cones. The filaments in the cone move forward relative to the substrate. (2) Actin turns over at a rate that is much slower than that of actin in lamellipodia and Listeria comet tails. In both of these structures, filaments turn over in 1-2 minutes, but in case of actin cones, turnover takes 12 minutes. (3). Another puzzle is the stability of actin cones to depolymerization by the actin depolymerizing drug latrunculin A (LTA). Actin completely turns over in 12 minutes in moving cones, so it might be expected that LTA would cause depolymerization in that time frame. However, even after 2 hours of LTA treatment, cones remain. It is likely that actin in cones is stabilized by binding of cross linkers or other proteins, but an understanding is not yet clear of the mechanism that regulates stability to permit turnover as the cones move, but prevents depolymerization when assembly stops (Noguchi, 2003).
Despite these differences from other motility processes, the favored model involves only actin assembly as the driving force for motility. In order to explain the dynamics of actin in the cone and results of pharmacological experiments, it is suggested that there are two actin structural components in a moving actin cone. The first actin structure is the actin cone itself. Three characteristics (stability, filament translocation and slow turnover) suggest that the actin cone is a highly organized and stable structure compared to the actin network in lamellipodia. It is likely that each actin cone moves forward as one unit. The second actin structure component is an actin network near the membrane that pushes the actin cone forward by force of polymerization. Actin filaments elongate near the membrane, similar to the leading edge, but the membrane is held rigid, rather than protruding as it does at the leading edge. In this case, a photobleached GFP-actin in a filament would be pushed away from the membrane, i.e. `forward' relative to the membrane. Eventually, this filament would be crosslinked into the actin cone as new actin filaments assembled. This model is consistent with the data, no information is available about the orientation of actin filaments and the sites of actin monomer incorporation that would provide additional support for such a model. In addition, the molecules that might be important to keep the membrane rigid and prevent its protrusion are as yet unknown (Noguchi, 2003).
An alternative model is that myosin based motility contributes to movement by generating force using cortical actin and/or actin cones as a substrate for movement. Using a motor protein to provide force is compatible with the idea that the actin cone moves forward as a unit. However, it is not obvious why motor-driven movement would be coupled with actin dynamics. Perhaps movement requires a dynamic cortical actin network around the actin cone, to provide tracks for myosin movement. This track might need to be continually assembled at new sites during movement. Since the actin cone is a very large and intensely labeled structure, it might be hard to detect the different dynamic behavior in a less prominent, thin cortical structure in the same region. Myosin VI is present on actin cones and essential for individualization, making it tempting to speculate that myosin VI might provide the driving force. However, cystic bulges of myosin VI mutants can move partway along the cyst, indicating its function is not required for cone movement. Instead, myosin VI is important for regulating actin dynamics during movement. Other myosins may be important for some aspects of cystic bulge movement, but this remains to be demonstrated. BDM, an inhibitor of myosin ATPases, did not block movement. This inhibitor has been demonstrated to block activity of myosin I, II and V, suggesting it is a general inhibitor. The lack of effect of BDM on cone movement makes a myosin-based motility model less likely: myosins cannot be ruled out as force generators in this process at this time. Mutant alleles of all the predicted myosins in the Drosophila are not available and the effect of BDM on myosins in many classes is unknown (Noguchi, 2003).
Microtubule-based motility is not likely to be involved in cone movement. There are no cytoplasmic microtubules, which might participate in generating force in cooperation with microtubule motors and inhibitors of microtubule dynamics, and motors do not stop movement. In addition, when actin dynamics are altered, the cystic bulge stops immediately. If the movement was microtubule based, it is not clear why actin dynamics would be important (Noguchi, 2003).
Further studies are required to provide support for this model of actin cone motility. Additional studies examining membrane dynamics, effect of disruption of actin polymerization regulators and ultrastructure of the actin cones will be needed for more insight into the similarities and differences in the mechanism of actin cone movement and lamellipodia extension (Noguchi, 2003).
Although the mechanism of actin cone movement is not fully understood, it is possible to speculate about the role of actin cones during individualization. It is suggested that the actin cones have three roles: (1) the actin cones have the ability to push the cystic bulge forward, using actin polymerization; (2) the actin cones sweep the cytoplasm and organelles out of the sperm flagella, acting as a sieve; (3) the actin cones must bind the cell membrane around them and shape it into the observed thin tubular structure. Eventually, as the actin depolymerizes at the cone tip, the membrane must attach to the axoneme (Noguchi, 2003).
There is an interesting transition that occurs as individualization begins. Microtubule staining disappears during a very short period around the onset of actin cone movement. The data suggests that this disappearance is due to tubulin degradation as movement begins. This idea is supported by observations that the amount of tubulin present in individualized spermatids is much less than in cysts prior to individualization, and that cytoplasmic microtubules disappear during individualization (Noguchi, 2003).
This transition temporally coincides with the onset of actin cone movement, rather than sperm nucleus DNA condensation. FRAP experiments demonstrate that actin dynamics also accelerate after the onset of movement. Therefore, it is suspected that a global signal orchestrates these events to trigger the onset of individualization (Noguchi, 2003).
Membrane remodeling does not require endocytosis or exocytosis. Conventional endocytosis may not be important for movement of the cystic bulge, because FM1-43 staining of cell membrane demonstrates that membrane uptake does not take place around the actin cones, and blocking endocytosis using temperature shift of the shibire (dynamin) mutant does not affect cystic bulge movement. In addition, no concentration of alpha-adaptin has been observed in the region around the actin cones, suggesting that no coated pit formation occurs there. Conversely, clathrin mutants have defects in individualization, but the reason that individualization fails has not been well studied. The discrepancies in these data will only be resolved by further analysis of the clathrin mutant phenotype and studies of the effects of loss of function in other proteins in the endocytosis pathway (Noguchi, 2003).
Likewise, exocytosis may not play a major role in the membrane remodeling process, because membrane staining with the dye FM1-43 suggests that there is not a significant amount of membrane insertion at the sites around the actin cones, and treatment with the the exocytosis inhibitor BFA does not affect the movement of cystic bulge. These data do not completely exclude the possibility that exocytic events participate in remodeling, since the exocytosis could not be directly measured. However, it seems more likely that the large number of membrane invaginations that are present in the cystic bulge is a sufficient source of membrane to accomplish remodeling. The plasma membrane seems to be smoothly reorganized into thin tubular structures around the actin cones. Furthermore, ultrastructural observations have shown that the membrane around actin cones is flat, without any invaginating or docking membrane vesicles. These data support the idea that the cell membrane in the cystic bulge is directly deformed into a thin tubular structure (Noguchi, 2003).
In animal cells, cytokinesis is accomplished by the contractile ring, a transient structure containing actin and myosin II filaments (Zipper) that is anchored to the equatorial cortex. Interactions between these filaments lead to the constriction of a ring that pinches the dividing cell in the middle like an ever tightening purse string until cleavage is completed. Male meiosis was examined in mutants of the chickadee (chic) locus, a Drosophila gene that encodes profilin, a low molecular weight actin-binding protein that modulates F-actin polymerization. These mutants are severely defective in meiotic cytokinesis. Difficulties in meiotic cytokinesis are immediately obvious because of the characteristic appearance of spermatids directly after their formation at the so-called onion stage. Wild-type onion stage spermatids contain a single phase-light nucleus and a similarly sized phase-dark Nebenkern (a mitochondrial derivative). Failures in cytokinesis result in abnormally large Nebenkern associated with multiple normal-sized nuclei. The resulting phenotypes fall into multiple groups: in testes of males homozygous for chic a large fraction of onion-stage spermatids contain a single Nebenkern of larger than normal size, associated with two or more normal-sized nuclei. A substantial proportion have two nuclei with an intermediate-size Nebekern, but most frequently, these aberrant spermatids contain four nuclei and a very large Nebenkern. These phenotypes reflect failures of cytokinesis at either one or the other or both meiotic divisions, respectively, which would prevent proper subdivision of mitochondria and nuclei into daughter spermatids (Giansanti, 1998).
In wild-type testes, phalloidin staining reveals an F-actin-enriched contractile ring that encircles the spindle midzone (the bundle of interdigitated microtubules between the separating chromosome complements during anaphase and telophase) from late anaphase through telophase of both meiotic divisions. In contrast, in many meiotic divisions of chic mutants, no actin staining is visible at the cell equator. In most mutant ana-telophases with equatorial actin staining, only irregular patches of F-actin are observed. Chic protein is found in clear concentrations near the cell cortex, particularly in the equatorial zone. In chic mutants abnormal aggregations of F-actin are observed primarily in premeiotic mature spermatocytes at the S5 stage. These aggregates are enriched in alpha-spectrin and are almost invariably associated with ring canals, suggesting that these actin aggregates are in fact relatively undegraded remnants of the male fusome (see Drosophila Spectrin for more information on fusomes). It is suggested that in the absence of chic the disintegration of the fusome is partially blocked (Giansanti, 1998).
In addition to the absence of the contractile ring, the ana-telophases of all the chic mutants exhibit another obvious abnormality: a defect in central spindle morphology. In wild-type ana-telophases of both meiotic divisions, the two daughter nuclei are connected by a prominent bundle of interdigitating microtubules. In chic mutants, this central spindle structure is considerably less dense than in wild type, and microtubules show very little or no interdigitation. In wild type, the central spindle is already evident by mid-anaphase, before the actin ring constriction has caused substantial equatorial pinching. In the strongest chic mutants, these early stages of central spindle formation are never seen, indicating that the chic phenotype reflects a failure of central spindle assembly rather than a degradation of this structure in the absence of the actin ring. This result is surprising because the central spindle is a tubulin based cytoskeletal structure. There is substantial evidence that the central spindle is required for structuring of the actin based contractile ring but not similar evidence that the contractile ring is required for building the central spindle. Together, these observations indicate that chic mutations disrupt two major cytokinetic structures: the microtubule-based central spindle and the actomyosin contractile ring (Giansanti, 1998).
In wild-type primary spermatocytes during the prophase-prometaphase transition of the first meiotic division (stage M1), centrosomes migrate from a position just under the plasma membrane to the nuclear envelope, where they nucleate prominent asters. The two asters then separate and move around the periphery of the nuclear membrane, so as to establish a bipolar spindle. Similarly, during late telophase of the first meiotic division and the short interphase between meiosis I and meiosis II, asters separate and migrate to the opposite poles of secondary spermatocytes. Although the asters in chic mutants are improperly positioned by the start of prometaphase, relatively normal-looking bipolar spindles eventually form by late metaphase. It is remarkable that the function of these spindles, with respect to chromosome segregation, appears to be largely unimpaired. Only a very low frequency of onion-stage spermatids contain irregularly sized nuclei, such as micronuclei (Giansanti, 1998).
Lesions in twinstar (tsr), a gene encoding a Drosophila cofilin (an actin severing and depolymerizing protein), cause a syndrome of phenotypic effects that have both similarities and differences to those described above for chic mutations. In tsr, as in chic spermatocytes, centrosome separation and migration are defective, abnormal accumulations of F-actin are apparent, and cytokinesis often fails after one or both meiotic divisions. tsr and chic mutant phenotypes, however, can be easily distinguished on the basis of several criteria. In tsr mutants, the central spindle is normal, and the contractile ring still forms (though in misshapen and enlarged form), in contrast to the situation for chic. The types of F-actin aggregates formed in tsr spermatocytes are much different from those in chic. It is concluded that tsr and chic mutations differ in how they interfere with meiosis (Giansanti, 1998).
To further investigate the relationships between the central spindle and the contractile ring, meiosis was examined in the cytokinesis-defective mutants KLP3A and diaphanous. The KLP3A gene encodes a kinesin-like protein that accumulates in the central spindle midzone during anaphase and telophase of both meiotic divisions. Accordingly, mutations in this gene disrupt central spindle formation and cause frequent failures in meiotic cytokinesis. To check whether the defect in central spindle integrity observed in KLP3A mutants also affects actin ring assembly, KLP3A mutant testes were stained with rhodamine-labeled phalloidin. The results of this experiment clearly show that most mutant ana-telophases (90%) are completely devoid of actin rings. The rare ana-telophases that exhibit thin and incomplete actin rings also contain more densely packed central spindles than those of cells completely lacking contractile rings. Despite the absence of the contractile ring, KLP3A mutants do not exhibit aberrant actin accumulations or problems in aster migration like those described above for chic and tsr mutants (Giansanti, 1998).
The diaphanous gene encodes a protein that interacts with profilin through its proline-rich domain. All the ana-telophases present in testes homozygous for dia mutants are completely devoid of actin rings. It is of interest that these figures also show severe defects in the central spindle, similar to those observed in chic and KLP3A. The effects on the actomyosin contractile ring and the central spindle observed in chic and dia mutants could be specific consequences of lesions in the corresponding gene products. Alternatively, these effects could result from a more general disruption of the actin cytoskeleton. To discriminate between these possibilities, wild-type testes were treated with cytochalasin B prior to fixation and staining. Cytochalasin B binds the barbed ends of actin filaments and promotes the conversion of ATP-actin monomers to ADP-actin , preventing proper assembly of the contractile ring in most cell types. Remarkably, incubation with this drug produces an almost exact phenocopy of strong chic alleles. No F-actin staining is observed in any contractile ring-like structures at the equator of ana-telophase cells. (Giansanti, 1998).
In all cases examined, the central spindle and the contractile ring in meiotic ana-telophases were simultaneously absent. Together, these results suggest a cooperative interaction between elements of the actin-based contractile ring and the central spindle microtubules: when one of these structures is disrupted, the proper assembly of the other is also affected. In addition to effects on the central spindle and the cytokinetic apparatus, another consequence of chic mutations was observed: A large fraction of chic spermatocytes exhibit abnormal positioning and delayed migration of asters to the cell poles. A similar phenotype was seen in testes treated with cytochalasin B and has been noted previously in mutants at the twinstar locus. These observations all indicate that proper actin assembly is necessary for centrosome separation and migration, and that the central spindle and the contractile ring are interdependent structures (Giansanti, 1998).
The best candidate at present for mediating interactions between the central spindle and cortical actin, at least during male meiosis, is the KLP3A kinesin-like protein. This protein could interact directly with both the central spindle microtubules and components of the contractile ring. Alternatively, KLP3A could transport to the spindle midzone molecules that mediate F actin-microtubule interactions. At the moment, it is not possible to discriminate between these possibilities, nor is there any information on the proteins that bind to or might be transported by KLP3A. It is believed, however, that the isolation and characterization of additional mutations causing cytological phenotypes similar to those of KLP3A, chic, and dia, will eventually provide substantial insight into the mechanisms underlying microtubule-actin interaction during cytokinesis (Giansanti, 1998).
The molecular identity and function of the Drosophila melanogaster Y-linked fertility factors have long eluded researchers. Although the D. melanogaster genome sequence has recently been completed, the fertility factors still are not identified, in part because of low cloning efficiency of heterochromatic Y sequences. A method for iterative BLAST searching has been used to assemble heterochromatic genes from shotgun assemblies, and kl-2 and kl-3 have been identified as 1-beta and gamma-dynein heavy chains, respectively. These conclusions are supported by formal genetics with X-Y translocation lines. Reverse transcription-PCR was successful in linking together unmapped sequence fragments from the whole-genome shotgun assembly, although some sequences were missing altogether from the shotgun effort and had to be generated de novo. A previously undescribed Y gene, polycystine-related (PRY), was also found. The closest paralogs of kl-2, kl-3, and PRY (and also of kl-5) are autosomal and not X-linked, suggesting that the evolution of the Drosophila Y chromosome has been driven by an accumulation of male-related genes arising de novo from the autosomes (Carvalho, 2000).
The discovery that the Y chromosome of Drosophila melanogaster contains genes essential only for male fertility dates back to the birth of Drosophila genetics and the theory of chromosomal inheritance. In 1929, Stern showed that these genes are localized in both the short (YS) and long (YL) arms of the Y chromosome, and in 1960, Brosseau used x-ray-induced mutations to identify seven complementation groups, two in YS (ks-1 and ks-2) and five in YL (kl-1 to kl-5). In 1981, Kennison obtained fertile X-Y translocation lines and used them to construct males with deletions in each of the fertility factors. With these lines, Kennison confirmed six of the seven fertility factors previously identified by Brosseau (kl-4 was not confirmed). The same lines allowed a more precise identification of the defects associated with the lack of each of the fertility factors. In particular, the lack of kl-3 or kl-5 causes the loss of the outer arm of the sperm tail axoneme, a structure known to contain the molecular motor protein dynein in other organisms. Indeed, in 1982, Goldstein showed that sperm from kl-3- and kl-5- (and also kl-2-) males lack three discrete high molecular weight proteins with mobility similar to dynein heavy chains of Chlamydomonas reinhardtii and proposed that these fertility factors are the structural genes of three different dynein heavy chain proteins. In 1993, Gepner and Hays sequenced part of kl-5 and showed that it encodes an axonemal beta-dynein heavy chain that is expressed in the testis (Carvalho, 2000 and references therein).
Axonemal dynein heavy chains are known to be responsible for the beating of flagella and cilia, which explains why kl mutants produce immotile sperm. There are several isoforms of axonemal dynein heavy chains (alpha, beta, gamma, 1beta, 1alpha, etc.) that associate to form the inner and outer arms of the axonemes. D. melanogaster has at least seven other dynein heavy chain genes, scattered in chromosomes X, 2, and 3 (Carvalho, 2000 and references therein).
Another important experimental breakthrough was the development of a method to discern banding patterns in Drosophila heterochromatin, which allowed the first detailed cytogenetic investigation of the Y chromosome. Gatti and Pimpinelli (1983) identified 25 heterochromatic bands on the Y and mapped the fertility factors to these bands. It became clear that some of the fertility factors, including kl-5, are unusually large [~3 megabase (Mb)]. The paradox of a conventional coding gene (e.g., kl-5), spread over a huge amount of DNA was solved by Bünemann and coworkers: in the kl-5 homolog of Drosophila hydei, some of the introns are gigantic (>1 Mb) and most likely account for the unusual size of the gene. These introns are composed of short repetitive sequences and satellite DNA. These key discoveries trace back to the extensive work on lampbrush Y chromosomes initiated by Meyer and coworkers in 1961 (Carvalho, 2000 and references therein).
As can be seen from the above summary, the progress on the identification of Y-linked genes has been very slow. This slow progress is mainly a consequence of the technical difficulties caused by the heterochromatic state of the Y chromosome, and most of the experimental breakthroughs mentioned above actually are ingenious ways to implement standard tools used for euchromatic genes in heterochromatin. The Y chromosome does not recombine during meiosis, preventing classical genetic mapping; this problem was solved by Kennison's lines. It does not undergo polytenization, making cytogenetic studies more difficult [solved by Gatti and Pimpinelli]. P element mutagenesis was also more difficult, because the common markers are often silenced when inserted in the Y, but now there are special P constructs that make it possible to overcome this limitation (Carvalho, 2000 and references therein).
The recent sequencing of the Drosophila genome might have yielded the final solution, but again the heterochromatic nature of the Y chromosome posed special difficulties. Most heterochromatin is composed of short repetitive sequences that are not stable in the vectors used in sequencing projects. Thus, despite comprising nearly 30% of the genome, heterochromatic sequences account for only 2% of the sequence reads. Furthermore, its repetitive nature does not allow the assembly of the individual sequence reads (~500 bp) into larger scaffolds, and these into complete chromosome arms. As a result, only 15 kb (a small portion of the kl-5 gene) have been assigned to the Y chromosome, whereas essentially all of the 120 Mb of the euchromatin have been assembled into chromosomes X, 2, 3, and 4. Besides these mapped sequences, 631 scaffolds (ranging from 1 kb to 64 kb, and totaling ~4 Mb of sequence) remain unmapped. These unmapped scaffolds most likely contain pieces of heterochromatic genes, including Y-linked ones (Carvalho, 2000 and references therein).
Of special interest in the study has been the inventive use of iterative BLAST searching for assembly of heterochromatic sequences. The unmapped Drosophila scaffolds (called "armU" in Celera's CD-ROM release of the Drosophila genome) were downloaded from ftp://ncbi.nlm.nih.gov/genbank/genomes/D_melanogaster/, and then an armU database was built by using the FORMATDB program of the STANDALONE BLAST. In this way BLAST searches could be restricted to the set of unmapped scaffolds. In addition to STANDALONE BLAST, extensive use was made of the programs WWWSTANDALONE BLAST (Linux version), NETBLAST, REPEATMASKER (available at http://repeatmasker.genome.washington.edu/cgi-bin/RepeatMasker), and NAP and GAP2 (available at http://genome.cs.mtu.edu/sas.html). BLAST programs were downloaded from the National Center of Biotechnology Information (http://www.ncbi.nlm.nih.gov/) (Carvalho, 2000 and references therein).
To see how a Y-linked gene appears in the armU sequences, the complete cDNA of kl-5 (Genbank nucleotide record: AF210453) was used as a query sequence in a BLASTN search against the armU database. In addition to the fragment already identified (CG17616 gene in the Genbank AE002688 scaffold), most of the kl-5 gene was retrieved, scattered across five scaffolds. The majority of these scaffolds contain complete exons (the exception is AE003233, which begins in the middle of an exon). Their 3' ends contain the 5' splice junctions and a variable portion of downstream intronic sequences, often ending with simple repetitive sequences. The 5' ends of scaffolds in armU have analogous structures. Some exons are missing altogether in armU. These observations fit well with the expected behavior of a gene like kl-5 in whole genome shotgun (WGS) projects: exons define unique, nonrepetitive sequences that will be cloned regularly and will be assembled into at least a small scaffold in the end of WGS. Normally sized introns will be readily cloned and assembled along with exons. Indeed, most of the kl-5 scaffolds contain several exons and the intervening short introns. However, some introns of kl-5 probably contain Mega base-sized blocks of repetitive DNA that cannot be assembled by WGS or any other available method. These fragments will rarely be cloned and sequenced and, even if sequenced, would not be assembled into a scaffold. In short, during WGS, a gene like kl-5 will be chopped into several pieces, delimited by the unclonable intronic satellite DNA. Most of the time, a gene immersed in heterochromatin will go undetected by the normal 'first pass' annotation procedures (which rely on gene prediction tools and BLASTX with high stringency), because these methods will work poorly with individual exon sequences. However, the whole gene may be retrieved if a suitable query sequence is available (the kl-5 cDNA in this case) to identify and align its pieces. Very small exons embedded in large introns will most likely be lost during the WGS, and this probably explains the lack of some kl-5 exons (Carvalho, 2000).
To identify other fertility factors in armU, protein sequences were used as query sequences. Suitable proteins were chosen as follows. First the 631 scaffolds of armU were filtered with REPEATMASKER and a BLASTX search of each of them was performed against the nr database (all known proteins, including putative ones) with a rather high stringency (e = 10-4). There were proteins that gave hits in hundreds of scaffolds; most of them are reverse transcriptases, copia polyprotein, etc., and most likely are matching transposable elements of armU that 'escaped' REPEATMASKER. Some other proteins have hits in a few scaffolds; these are homologs of prospective Y-linked genes, chopped in pieces as kl-5. Each of these prospective proteins was used as a query sequence (as was done with the cDNA of kl-5), running TBLASTN with a lower stringency (e = 10) against the armU database. A staggered pattern results from the very large introns of Y-linked genes. Two such cases are myosin VII (AAF06035, from Dictyostelium discoideum) and gamma-dynein heavy chain, from C. reinhardtii], and were investigated further. All tested scaffolds relating to myosin VII proved to be not Y-linked (i.e., PCR produces bands when either male or female DNA is used as the template), whereas most of the dynein-related were Y-linked. Several of the Y scaffolds were identified by using gamma-dynein heavy chain (Genbank record: Q39575) as a TBLASTN query sequence with a low stringency (sometimes e = 1,000), in an attempt to retrieve missing exons. Because there is a big overlap among several of the dynein-related scaffolds, most likely two different Y-linked dynein heavy chain genes were found (Carvalho, 2000).
Genomic DNA from Y deficient males (kl-1-, kl-2-, etc.) was used in PCR to map each of the Y-linked scaffolds identified in the previous step. It should be noted that this procedure assigns a given scaffold to a region of the Y chromosome (e.g., the kl-2 region), but it does not necessarily imply that this scaffold belongs to the actual fertility gene. This distinction is important, because a given region may contain more than one gene. For the sake of simplicity the regions of the Y are referred to by the name of the respective fertility factor they carry (Carvalho, 2000).
Several exons of kl-2 and kl-3 genes are missing from the BLAST results. These missing exons may be absent in armU sequences (as happened with kl-5) or may have diverged enough to be no longer identified by these methods. RT-PCR was used to obtain the sequence of these missing exons and to check whether the Y sequences that were detected are expressed. RT-PCR sequences were obtained from all splice junctions between adjacent scaffolds so that they could be precisely identified. The sequencing of the gaps revealed several previously missed armU scaffolds. In kl-2, some 330 codons of the N terminus are still missing. AE003086 filled the gap between AE003157 and AE002962. There is no sequence gap between AE002962 and AE003049. AE002706 filled a small portion of the gap between AE003049 and AE003219; the remaining 2 kb were sequenced and it was found that it is entirely missing in armU. The AE003219 scaffold contains five internal, short introns and extends through the stop codon. Regarding kl-3, AE002577 and AE002776 appear to be spurious matches caused by running TBLASTN with low stringency, because no RT-PCR product could be recovered that includes these sequences. The gap between AE002917 and AE002920 was sequenced; the 948-bp sequence is missing in armU. Finally, some 230 codons in the C terminus seem to be missing. Each of the three big kl-3 scaffolds contains one internal intron (Carvalho, 2000).
The internal introns were identified and localized with the NAP program, which aligns genomic DNA with proteins allowing for GT/AG bounded gaps (in the case presented here, armU scaffolds and the gamma-dynein Genbank sequence Q39575 were aligned). RT-PCR sequences surrounding each putative intron were obtained and aligned with the corresponding armU scaffold with the GAP2 program, which aligns genomic DNA with cDNA, again allowing for GT/AG bounded gaps. Almost all putative introns suggested by NAP were confirmed, although the inferred splice junctions frequently were not precise. Seven frame-shift sequence errors in armU sequences were pinpointed by NAP and BLASTX and were corrected by sequencing (Carvalho, 2000).
The assembled cDNA of kl-2 and kl-3 were deposited in GenBank under the accession numbers AF313479 and AF313480. Polycystine-related (PRY) is a putative, previously unidentified Y-linked gene. During attempts to retrieve missing exons two armU scaffolds were found that map to the kl-5 region but have no similarity with the kl-5 cDNA. AE002774 seems to contain only two short pieces of transposable elements and was not further investigated. AE003011 showed a strong similarity with the product of a putative gene localized in chromosome 2 (AAF44887) and also a weaker similarity with the human polycystine protein (AAD18021). Interestingly, polycystine is similar to the sea urchin sperm receptor for egg jelly (AAB08448). Using the Drosophila hypothetical protein AAF44887 as a query sequence in TBLASTN (against armU sequences) another closely related scaffold, AE003212 was recovered. RT-PCR closed the gap between it and AE003011; thus, they most likely are part of a previously unidentified expressed Y-linked gene, which is currently being sequenced. Surprisingly, AE003212 maps to the kl-3 region. These findings imply that the breakpoint of the V24 translocation (the h4 band) cuts the PRY gene in the middle. Thus, V24 is defective for PRY -- a close examination of this line may give some clue about the function of this gene. Because the kl-3 and kl-5 regions are known to contain factors (other than the dyneins) that cause sterility when present in three copies, it is possible that PRY is responsible for this phenotype (Carvalho, 2000).
A simple method has been described for tailoring BLAST searches in such a way that poorly assembled fragments from WGS projects may reveal genes embedded in heterochromatin. The method relies on TBLASTN searches (instead of the more usual BLASTX) to identify putative heterochromatic genes by the distinct staggered pattern they produce. It was applied and the kl-2 and kl-3 genes on the Y chromosome of D. melanogaster were successfully identified. The sequences that were found are long, transcribed open reading frames that encode dynein heavy chain polypeptides. Thus the quest for the Y dyneins initiated by Hardy is now finished, and Drosophila Y gene hunting may now move to the even more exciting task of identifying the mysterious kl-1, ks-1, and ks-2 (Carvalho, 2000).
Heterochromatic genes are islands of unique sequence and appear in the end of WGS as isolated scaffolds that could not be assembled into chromosomes. If introns are large enough and contain heterochromatic repeat sequences, they will be sufficiently underrepresented in the WGS sequences to disrupt the assembly of flanking scaffolds. As a result, exons of the same gene are scattered in several unmapped scaffolds ('armU'), generating a staggered pattern in TBLASTN and BLASTN searches. This pattern will occur not only in the Y-linked genes but also in autosomal heterochromatin, as in the case of the Drosophila myosin VII homolog. Although heterochromatic genes pose special problems for genome sequencing, it is possible that they have an advantage over euchromatic genes: once the euchromatic sequence of a whole chromosome arm (a Mb-sized, unique sequence scaffold) is obtained, it may be very difficult to detect all of the genes it contains, whereas it is possible that a significant proportion of the unique heterochromatic sequences represents functional genes. It seems that only functional sequences resist the forces that fill heterochromatic regions with short repeats and thus remain clonable, unique sequences. During this project, 38 armU scaffolds were examined, and at least 12 of them (~1/3) seem to be part of genes. Of course, this sample is not random, but it strongly suggests that the small isolated scaffolds remaining at the end of WGS projects may be a good source of interesting genes. Indeed, it is an advantage of the WGS approach over clone-based strategies that, besides the euchromatin, it also retrieves unique sequence heterochromatin, no matter how deeply the sequence is located within the heterochromatin (Carvalho, 2000).
A striking pattern emerges from the phylogeny of the Y dyneins: they all are closely related to other Drosophila genes, but none of these paralogous genes is X-linked. The same pattern occurs with PRY. Furthermore, the Drosophila X chromosome contains only one dynein heavy chain (Dhc 16F), in contrast with the three Y-linked ones. Thus, it seems likely that these genes were acquired from autosomes, rather than being present in the hypothetical chromosome pair that gave rise to the X and Y. This mechanism has been demonstrated for the mammalian Y, but in that case the Y chromosome also exhibits a number of X-derived genes. Another Drosophila Y-linked gene, Su(Ste), has been shown to be recently originated from an autosomal gene. Repetitive sequences also do not show any sign of X-Y homology; it has been proposed that much of the Drosophila Y is virtually a new construct, rather than a degenerated X. The data presented in this study clearly support this hypothesis. It remains to be seen whether any part of the ancestral Y was homologous to the X (as may be the case for rDNA genes, which are present in both X and Y chromosomes) or whether it is a totally new construct. This picture of the Drosophila Y may change if other, yet unidentified Y genes (kl-1, ks-1, ks-2, etc.) turn out to have X homologs. Whatever its origin, the present configuration of the Drosophila Y chromosome seems to be quite old, for at least kl-5 is present also in D. hydei and Drosophila mediopunctata, which diverged from D. melanogaster ~39 million years ago. A few Drosophila species have fertile X0 males; it will be most interesting to study the location of their axonemal dynein heavy chain genes (Carvalho, 2000).
The absence of X homologs and the close similarity between Y and autosomal genes suggest that the former is an agglomeration of autosomal genes. This hypothesis is the most parsimonious and explains well the kl-5, kl-3, and PRY cases. However, it is also possible that Y chromosomal genes have transposed to the autosomes, and this possibility might explain the kl-2 case. The closest paralogs of kl-3 and kl-2 are the CG9492 and CG9068 genes, respectively. Dynein heavy chains have ~4,500 amino acids, whereas CG9492 and CG9068 are shorter (3,508 and 1,227, respectively) and seem to lack the C terminus. The former case results from a misannotation: BLASTX and NAP identified all of the missing ~1,000 amino acids of CG9492 (including the stop codon at position 188,944 in the AE003683 scaffold). However, CG9068 seems to be truncated, for no sign of the 'missing' C terminus could be found. Therefore, the relationship between CG9068 and kl-2 is unclear; it is possible that kl-2 originated from CG9068 and that after this the latter suffered a deletion. However, it is also possible that CG9068 results from a partial transposition (perhaps being a pseudogene) of kl-2 (Carvalho, 2000).
The phylogeny of the dyneins strongly suggests that kl-2 encodes a 1beta-dynein, whereas kl-3 encodes a gamma-dynein. This phylogeny fits well with the known mutant phenotypes of kl genes and with the function of dynein heavy chains; kl-3- mutations (but not kl-2-) disrupt the outer arms of axonemal microtubules, and gamma-dyneins are part of these structures. 1beta-dyneins are part of the inner arms, and it remains to be explained why kl-2- mutants do not show cytological defects (Carvalho, 2000).
It has been noted that the human Y chromosome exhibits a 'functional coherence'; besides housekeeping genes, many Y genes have male-related functions, which contrasts with the random content of the other chromosomes. It is striking that the Drosophila Y has an even stronger coherence, approaching obsession; all known fertility factors (kl-2, kl-3, and kl-5) encode proteins belonging to the same gene family (axonemal dynein heavy chain). This extreme functional coherence, coupled with the lack of X homologs (which might provide an 'historical' cause), begs for an explanation (Carvalho, 2000).
Theoretically, the Y chromosome is expected to accumulate male-related genes; male-female antagonistic effect of genes may hamper the evolution of male-related traits, unless they are located in a male-specific region of the genome. This prediction has been demonstrated experimentally, and the findings presented here support it. Regarding the particular male fitness trait involved, the most likely advantage conferred by sperm axonemal motor proteins is sperm competitive ability. The PRY gene may also be involved in sperm competition if it has a function similar to its homolog in sea urchin. Drosophila females mate several times; thus, there is ample room for sperm competition, and clearly there is genetic variation for this trait. It is proposed that the evolution of the Drosophila Y chromosome has been driven by an accumulation of male-related genes, most likely caused by sperm competition. This hypothesis explains the puzzling finding of a Y chromosome packed with motor proteins which are absent in the X chromosome. The large element of chance involved in the occurrence of the appropriate translocations probably explains the apparent incompleteness of the process, that is, outer arms are composed of alpha-, beta-, and gamma-dyneins, but only beta and gamma got Y counterparts (Carvalho, 2000).
The hypothesis that natural selection has driven an accumulation on the
Drosophila Y of genes related to sperm function may be
tested in several ways. (1) Studies designed to quantify Y-linked
variation in sperm competition are clearly needed. (2) The comparative
method of looking for dynein heavy chain genes in other Diptera
(including species with fertile X0 males) may reveal the intermediate
steps of the birth of dynein-packed Y chromosomes. (3) the
identification of the other fertility factors may yield more clues
about the forces shaping Y chromosome evolution in
Drosophila (Carvalho, 2000).
Chromatin condensation is a typical feature of sperm cells. During mammalian
spermiogenesis, histones are first replaced by transition proteins and then by
protamines, while little of this process is known for Drosophila. This study characterizes
three genes in the fly genome, Mst35Ba, Mst35Bb, and
Mst77F. The results indicate that Mst35Ba and Mst35Bb
encode dProtA and dProtB, respectively. These are considerably larger than
mammalian protamines, but, as in mammals, both protamines contain typical
cysteine/arginine clusters. Mst77F encodes a linker histone-like protein
showing significant similarity to mammalian HILS1 protein. ProtamineA-enhanced
green fluorescent protein (eGFP), ProtamineB-eGFP, and Mst77F-eGFP carrying
Drosophila lines show that these proteins become the important
chromosomal protein components of elongating spermatids, and His2AvDGFP
vanishes. Mst77F mutants [ms(3)nc3] are
characterized by small round nuclei and are sterile as males. These data suggest
the major features of chromatin condensation in Drosophila
spermatogenesis correspond to those in mammals. During early fertilization
steps, the paternal pronucleus still contains protamines and Mst77F but regains
a nucleosomal conformation before zygote formation. In eggs laid by
sesame-deficient females, the paternal pronucleus remains in a
protamine-based chromatin status but Mst77F-eGFP is removed, suggesting that the
sesame gene product is essential for removal of protamines while Mst77F
removal is independent of Sesame (Raja, 2005).
For mammals, the somatic set of histones are modified, as these
are in part replaced by specific variants during meiotic prophase. After
meiosis, histones are replaced by major transition proteins TP1 and TP2
and subsequently by highly basic protamines to ensure the
remodeling of chromatin to a typically highly condensed and transcriptionally
silent state of mature sperm. These replacements leads to a shift from
histone-based nucleosomal conformation to a radically different conformation,
resembling stacked doughnut structures containing protamines as major chromatin
condensing proteins and DNA. Some mammals have only one protamine gene,
while mice and humans have two genes encoding two
different protamines, both of which are essential for fertility and are
haploinsufficient. HILS1 (spermatid-specific
linker histone H1-like protein) has been proposed to participate in chromatin
remodeling in mouse and human spermiogenesis.
The transition between histone removal and its replacement
by protamines in mice and humans is characterized by small 6- to 10-kDa
transition proteins acting as a short-term chromosomal proteins.
In mice, the transition proteins TP1 and TP2 are redundant
in function. In fishes and birds, transition proteins are missing and protamines
directly reorganize the chromatin. In annelids and echinoderms, the nucleosomal
configuration is maintained in sperms,
while protamine-like proteins have been described for
mussels. These protamine-like proteins lack the typical
high cysteine content necessary for disulfide bridges.
Therefore, a doughnut-type chromatin structure as in mammals is unlikely to
occur in mussels. It has been proposed that
the protamine-like proteins in mussels belong to the histone H1 family. The
sperm chromatin of mussels contain core histones and thus a nucleosomal
configuration, but histone H1 is replaced by protamine-like molecules which
organize the higher order structure of the chromatin (Raja, 2005).
For Drosophila melanogaster, chromatin reorganization after meiosis has
not been studied at the molecular level. At the light microscopic level,
the Drosophila spermatid nucleus is initially round after meiosis and
then is shaped to a thin needle-like structure with highly condensed chromatin,
so that the volume of the nucleus is condensed over 200-fold.
In mammals, the volume of the nucleus is reduced over
20-fold. In the mature sperms of Drosophila, core
histones are not detectable by immunohistology. There is
histochemical evidence for the presence of very basic proteins in sperm,
but it still remains an open question whether histones are
replaced by protamine-like basic proteins in Drosophila. The analysis of
the Drosophila genome sequence
revealed that the proteins encoded by two genes show similarity to mammalian
protamines for which the male-specific transcripts Mst35Ba and
Mst35Bb have been found and have been proposed to
encode protamine-like proteins. Another male specifically
transcribed gene, Mst77F, is a distant relative of the histone H1/H5
(linker histone) family and has been proposed to play a role either as a
transition protein or as a replacement protein for compaction of the
Drosophila sperm chromatin. With enhanced green
fluorescent protein (eGFP) fusion for these abovementioned proteins, this study shows that Mst35Ba and Mst35Bb indeed encode protamines and
Mst77F encodes a linker histone-like protein. The expression pattern of
Mst77F overlaps the pattern of protamines as a chromatin component. Furthermore, during fertilization, the removal of protamines from the male
pronucleus requires the function of the maternal component, Sesame, but not for
the removal of Mst77F. It has been shown that sesame mutants cause
impairment of the entry of histones into the male pronucleus (Raja, 2005).
Mst35Ba and Mst35Bb are
present at cytological position 35B6 and 35B6-7, respectively, on the chromosome
arm 2L. These two genes are arranged in tandem, and both consist of three exons.
The 5'UTR, coding region, and the 3'UTR of these
genes are highly identical; they probably arose from a
recent gene duplication. The encoded protamines show over 94% identity to each
other (Raja, 2005).
A remarkable feature of protamines is their ability to form intermolecular
disulfide bridges, which is reflected by the conserved cysteine residues within
mammalian protamines. The dProtA and dProtB are of 146
amino acids (aa) and 144 aa, respectively, and thus longer than even the human
and mouse Protamine-2, which are 102 aa and 107 aa, respectively.
Both Drosophila protamines contain 10 cysteines each
and show significant similarity, particularly with respect to a high cysteine,
lysine, and arginine content to mammalian protamines.
Human and mouse Protamine-1 aligns to the
N-terminal half of the Drosophila protamines (from aa positions 27 to
82), and four cysteine residues are conserved and regularly spaced. In contrast,
Protamine-2 of human and mouse
shows relatively high similarity to the C-terminal half of the Drosophila
protamines, with four cysteines in this region that are conserved and regularly
spaced, whereas one cysteine is shared with the mouse and human Protamine-1 (Raja, 2005).
Mst77F is present at the cytological position 77F on the chromosome arm 3L and
lies within the large intron of PKA-R1. Mst77F is also male specifically
transcribed, and the encoded protein has been proposed to be a linker histone
H1/H5 type, which could also play the role of a transition protein or a
protamine. The Mst77F protein shares a
significant similarity to the HILS1 protein of mouse
and human HILS1, where the percentages of cysteine,
lysine, and arginine are similar to that of mHILS1 and hHILS1.
HILS1 protein has been recently described as a component of
the mammalian sperm nucleus. Drosophila Mst77F
encodes a protein of 215 aa with a molecular mass of 24.5 kDa and with a pI of
9.86. mHILS1 is of 170 aa and shows 39% similarity to Mst77F.
Mst77F contains 10 cystine residues as in Drosophila
protamines, and mHILS1 contains eight cystine
residues, of which four residues are conserved (Raja, 2005).
As there are considerable differences between the mammalian protamines as well
as between the mammalian HILS1 proteins and the presumptive Drosophila
homologue Mst77F, additional experiments are essential to clarify if these
proteins are indeed involved in the condensation of sperm chromatin (Raja,
2005).
Drosophila protamine mRNAs are transcribed at the primary spermatocyte
stage, whereas in mammals protamine mRNAs are synthesized at the round
spermatid stage and translationally repressed until the
elongated stage, which is mediated by 3'UTR. The Drosophila
ProtamineA-eGFP and ProtamineB-eGFP constructs do not contain the 3'UTR of the
respective protamine genes. Nevertheless, the transgenic flies carrying
these constructs still show repression of translation. So, in Drosophila,
the region responsible for the translational repression is most likely in the
5'UTR. Deletion constructs of Mst35Bb and Mst77F 5'UTRs fused to
the reporter lacZ show that the translation repression element is indeed
present in the 5'UTR. This holds true also for the mRNA of the
Mst77F-eGFP fusion gene, as is the case for all mRNAs investigated
concerning translational repression so far in male germ lines of
Drosophila. In contrast to mammalian spermatogenesis, in Drosophila transcription
ceases already with the entry into meiotic divisions.
Since the protamines are made in the elongated spermatids, the transcriptional
silencing in Drosophila spermatogenesis seems to be independent of
protamines (Raja, 2005).
When primary amino acid sequences of Drosophila protamines are compared
to mammalian protamines, it is quite evident that Drosophila protamines
are relatively large. dProtA and dProtB are over 94% identical to each other.
This could explain that both the protamines may be functionally redundant. Human
and mouse Protamine-1 aligns with the N terminus of both Drosophila
protamines, and Protamine-2 aligns more to the C
terminus. It is possible that the Drosophila
protamines undergo posttranslational cleavage at the N terminus, as is known for
mammals. The cytoplasmic eGFP fused at the C terminus
shows clear nuclear localization, indicating that the tagged protamine is
functionally intact. Drosophila protamines each contain 10 cysteine
residues at identical positions, while over 4 of 10 cysteines at the N terminus
and the C terminus are conserved with human and mouse Protamine-1 and
Protamine-2, respectively. With
nine cysteines, the content is highest in Protamine-1 of mice. Inter- or
intra-disulfide bridges can be formed between the cysteine-rich protamines to
condense the DNA. For mice it is shown that mutation in
protamine-1 or protamine-2 is haploinsufficient and causes male
sterility. A haploid situation was analyzed for the Mst35Ba and
Mst35Bb genes with the deficiency Df(2L)Exel8033/+;
these flies are fertile males and show normal
spermatogenesis. The large amount of identity that both dProtA
and dProtB exhibit can contribute to the functional redundancy (Raja, 2005).
Chromatin reorganization is an essential feature during spermiogenesis. The
functional significance of chromatin compaction during spermiogenesis is still
unknown. The main explanation seems to be that compaction of the sperm nucleus
is an essential factor for its mobility as well as for the penetration of sperm
into the egg and genomic stability. In mammals, somatic histones are in part
replaced by spermatid-specific variants during meiotic prophase,
later by major transition proteins TP1 and TP2,
and subsequently by highly basic protamines to ensure the
remodeling of chromatin to a typically highly condensed and transcriptionally
silent state of mature sperm. These replacements lead to a shift from
histone-based nucleosomal conformation to a radically different conformation,
resembling stacked doughnut structures containing major chromatin condensing
proteins and DNA in the nucleus (Raja, 2005).
In Drosophila, so far no proteins have been identified that are involved
in the packaging of the genome in the mature sperm nucleus. One observation,
that Histone3.3 variant and the somatic H3 isoform in Drosophila are
vanishing at the time of chromatin condensation, supports the view of histone
displacement, but it was still a
question of whether it is the real absence of histones at this stage in
Drosophila or whether the antibodies are not accessible to the mature
sperm due to the tight packaging of the chromatin. To
circumvent this problem, the GFP fusion approach was chosen, use was made of the
existing His2AvDGFP, and
Protamine-eGFP and Mst77F-eGFP fusion transgenic flies were generated
in order to
analyze the situation in Drosophila. The results clearly show that
histone His2AvD is lost from the spermatid nuclei at the time of appearance of
protamines and Mst77F during later stages of spermatid differentiation. The
exact molecular mechanisms underlying the histone displacement, degradation, and
incorporation of protamines onto the chromatin are poorly understood.
For mammals, evidence has been obtained that histone H2A is
ubiquitinated in mouse spermatids around the developmental time period when
histones are removed from the chromatin.
The mammalian HR6B ubiquitin-conjugating enzyme is the
homologue of yeast RAD6, and both can ubiquitinate histones in vitro.
Thus far, the mechanism of histone displacement and protamine
incorporation is unknown during spermiogenesis in Drosophila. In
flies as well as in mammals, many questions remain unanswered that need
to be addressed about these
underlying mechanisms of chromatin remodeling during spermiogenesis (Raja, 2005).
In mammals, transition proteins act as intermediates in the histone-to-protamine
transition. In mice, the onset of HILS1 and transition
proteins TP1 and TP2 (major forms) overlaps with the pattern of Protamine-1 and
later with Protamine-2 but HILS1 and the transition proteins are no longer present in the mature sperm.
Mice lacking both TP1 and TP2 show normal transcriptional
repression, histone displacement, nuclear shaping, and protamine deposition but
show the loss of genomic integrity with large numbers of DNA breaks leading to
male sterility. In Drosophila,
histones are displaced with synchronous accumulation of protamines and Mst77F.
Mst77F, a distant relative of the histone H1/H5 (linker histone) family, has
been proposed to play a role either as a transition protein or as a protamine
for compaction of the Drosophila sperm chromatin.
Mst77F shows highest similarity to HILS1 with respect to the cysteines and basic
amino acid content but
not to mouse TP1, TP2, or H1t. Moreover, the results
show that the pattern of expression of Mst77F in the nucleus is similar to that
of mHILS1 in the nucleus, with the exception that Mst77F is also transiently
detected in the flagella and persists in mature sperm nuclei, unlike mHILS1. In
mammalian mature sperm nuclei, it is only the protamines that are the chromatin
condensing proteins which persist. This again raises the question of whether
Mst77F could also play the role of protamines. However, one additional copy of
dProtB (dProtA and dProtB showing 94% identity may be functionally redundant)
does not rescue the ms(3)nc3 phenotype, indicating that the
role of Mst77F may be completely or partially different from that of protamines
in the nucleus. However, a null mutation for Mst77F is required to answer
this question with respect to chromatin condensation. In
ms(3)nc3 mutants, the chromatin condensation with the
native protamines continues to take place. When a closer look was taken at the
deposition of ProtamineB-eGFP in
ms(3)nc3/Df(3L)ri-79c
trans-heterozygotes, it revealed that the condensed chromatin in the
tid-shaped nuclei is concentrated at the two
opposite ends, with a lightly stained chromatin spaced in the center. So the
chromatin condensation takes place but may not be complete with the
incorporation of the mutant Mst77F protein. The large amount of chromatin
compaction or condensation seen in Drosophila mature sperm when compared
to that of mouse and human sperm possibly could be the result of persistence of
Mst77F in the mature sperm nuclei. It remains to be clarified whether the sperm
nucleus contains further protamines that have not yet been properly annotated (Raja, 2005).
ms(3)nc3 is a second-site noncomplementation (nc) mutation
that was isolated in an ethylmethanesulfonate screen to identify interacting
proteins involved in microtubule function in Drosophila. This study shows
that ms(3)nc3 is a
single missense mutation from a T>A transition, causing the substitution of threonine instead of serine
at aa position 149. Mst77F shows a pattern of
expression similar to protamines in the nucleus and was also seen in the flagella until the
individualization stage. Since
ms(3)nc3 fails to complement class I alleles at the
ß2 tubulin locus, it is possible that Mst77F
has a dual role to play as a chromatin condensing protein in the nucleus and for
the normal nuclear shaping. Nuclear shaping is a microtubule-based event.
ms(3)nc3 leads to a tid-shaped nuclear
phenotype, where the nucleus fails to shape into a needle-like nuclei. Similar
defective nuclear shaping is seen with the few homozygous and heteroallelic
combinations of class I alleles of ß2 tubulin. The
incorporation of the defective subunit encoded by ms(3)nc3
may interfere with the function of the resulting complex. These data suggest the
involvement of an Mst77F (a linker histone variant) in the microtubule dynamics
during the nuclear shaping. This again complements the role of sea urchin
histone H1 in the stabilization of flagellar microtubules (Raja, 2005).
After the first steps in the fertilization process, the male gamete is still in
the highly compact protamine-based chromatin structure. In a wild-type egg, the
paternal pronucleus changes the shape from the needle-like to a spherical
structure. Furthermore, the male pronucleus acquires a nucleosome-based
structure before zygote formation and thus is transformed into a
replication-competent male pronucleus. sesame is a maternal effect
mutation in HIRA and had been mapped to 7C1.
HIRA family of genes (named after yeast HIR genes; HIR is an acronym for
'histone regulator') includes the yeast HIR1 and HIR2 repressors of histone gene
transcription in S. cerevisiae, human TUPLE-1/HIRA, chicken HIRA, and mouse HIRA. In
Drosophila, HIRA is expressed in the female germ line and a high level of
HIRA mRNA is deposited in the egg. Human HIRA is
shown to bind to histone H2B and H4. The WD repeats
present at the N-terminal part of HIRA could probably function as a part of a
multiprotein complex. Xenopus HIRA proteins are
also known in promoting chromatin assembly that is independent of DNA synthesis
in vitro. The corresponding maternal effect mutant sesame, in which the sperm fertilizes the egg but no zygote is formed, has been analyzed. Although the shape change of the nucleus to the spherical structure occurs in these mutants, maternal histones are not incorporated into the male pronucleus, which
strengthens the function of HIRA in binding to the core histones. This study shows
that neither Drosophila protamine is removed from the male pronucleus
in sesame mutants. This leads to the proposal that the transport and
incorporation of histones onto the chromatin in some manner is coupled to the
removal of protamines in which HIRA could play an important role in the
multiprotein complex required in this chromatin reconstitution process.
Mst77F removal from the male pronucleus in contrast to protamines
is independent of HIRA (Raja, 2005).
During spermiogenesis, chromatin reorganization of the complete genome is an
essential feature for male fertility. This process leads to an extremely
condensed state of the haploid genome in the sperm and requires a reorganization
of the paternal genome in the male pronucleus during fertilization and before
zygote formation. With the characterization of the chromatin condensing proteins
in Drosophila, it would be possible to gain more insight into the
mechanisms of sperm chromatin reorganization during spermiogenesis and
fertilization (Raja, 2005).
In higher organisms, the chromatin of sperm is organised in a highly condensed protamine-based structure. In pre-meiotic stages and shortly after meiosis, histones carry multiple modifications. This study focused on post-meiotic stages and shows that also after meiosis, histone H3 shows a high overall methylation of K9 and K27; it was hypothesised that these modifications ensure maintenance of transcriptional silencing in the haploid genome. Furthermore, histones are lost during the early canoe stage, and just before this stage, hyper-acetylation of histone H4 and mono-ubiquitylation of histone H2A occurs. It is believed that these histone modifications within the histone-based chromatin architecture may lead to better access of enzymes and chromatin remodellers. This notion is supported by the presence of the architectural protein CTCF, numerous DNA breaks, SUMO, UbcD6 and high content of ubiquitin, as well as testes-specific nuclear proteasomes at this time. Moreover, the first transition protein-like chromosomal protein to be found in Drosophila, Tpl94D, is reported. It is proposed that Tpl94D (an HMG box protein) and the numerous DNA breaks facilitate chromatin unwinding as a prelude to protamine and Mst77F deposition. Finally, it is showm that histone modifications and removal are independent of protamine synthesis (Rathke, 2007).
The switch between a nucleosome-based chromatin configuration and a protamine-based structure is a specialised form of chromatin remodelling in the male germline. The mammalian zinc finger protein CTCF is involved in many epigenetic processes. Furthermore, paralogous variant of CTCF which is testis-specifically expressed, called BORIS, is exclusively expressed in the mammalian male germline. The function of BORIS in this context is still not clear. Drosophila, in contrast to mammals, contains only one CTCF gene. It was therefore asked whether Drosophila CTCF is also expressed in the testes, and immunostaining and anti-histone staining was performed on testes of transgenic flies expressing protamine-eGFP. CTCF expression was observed during pre-meiotic and meiotic stages at the chromosomes as has been shown for mitotic cell division in mammalian cell culture. Shortly after meiosis, CTCF is visible in young elongating nuclei, where it co-localises with the chromatin as indicated by the histone distribution. CTCF is also present in the early and late canoe stage spermatid heads. At the early canoe stage, CTCF is very diffusely distributed in comparison to histones. CTCF does not co-localise with the chromatin which starts to condense at one side of the nucleus. This diffuse distribution is still visible at the late canoe stage when protamine-eGFP starts to be deposited to the chromatin. CTCF is no longer detectable after the canoe stage. The earlier chromatin-associated CTCF localisation might indicate a very early role in chromatin reorganisation at the switch between the nebenkern and canoe stage. Furthermore, CTCF might be associated primarily with the chromatin, which is not yet condensing during these stages. The late canoe stage is the only post-meiotic stage where distinct regions of RNA polymerase II are found with an antibody directed against a phosphorylated subunit of active polymerase, indicative of transcription. At this precise stage, only a very small set of genes is thought to be transcribed. Also CTCF expression during chromatin reorganisation in the nucleus was detected in D. hydei (Rathke, 2007).
Sperm morphogenesis is characterised by an impressive degree of changes in cell architecture based on stored, translationally repressed mRNAs that are recruited at the appropriate time to the polysomes. Among these are mRNAs that encode Tpl94D and protamines. A dramatic switch in structure from the nucleosomal- to the protamine-based structure of chromatin takes place, and this remarkable chromatin reorganisation of the complete genome is a typical feature depending on stored mRNAs, e.g. for protamine synthesis. This process ultimately leads to an extremely condensed state of the haploid genome in the sperm, which is essential for male fertility in mammals. This study focused on the switch between a nucleosomal- and a protamine-based chromatin reorganisation. The major steps in chromatin organisation take place in the canoe stage of spermatid development. A candidate for a transition protein in Drosophila was identified. The corresponding gene tpl94D (CG31281) encodes a predicted basic high mobility group (HMG) protein of 18.8 kDa. In transgenic flies, Tpl94D-eGFP fusion proteins are expressed solely during the switch between histones and protamines, as is typical for mammalian transition proteins. Since a highly similar chain of events to those reported in mammals is observed, the Drosophila system is considered an excellent choice to study the mechanism of chromatin remodelling during male germ cell development (Rathke, 2007).
Generally, the bulk of histones, including their diverse modifications in the N-terminal tail, appear to be removed during the canoe stage. Furthermore, the nucleus accumulates ubiquitin at the early canoe stage, when mono-ubiquitylation of histone H2A is no longer detectable. Therefore, taking into account the known presence of proteasomes in the nucleus at this stage of chromatin reorganisation and the overlap of expression shown in this study, it is hypothesised that this ubiquitylation is targeting histones for degradation. This study investigated several mutants having mutations in ubiquitin-conjugating enzymes or ubiquitin ligases, exhibiting arrested spermiogenesis during spermatid development and that are male sterile. However, in all investigated mutants, histone removal is indistinguishable from that of wild-type flies (Rathke, 2007).
Many histone modifications were found after meiosis and were categorised into three classes (Rathke, 2007).
It remains to be clarified whether and how these histone modifications influence the topology of the chromatin as a prelude to histone removal as well as for Tpl94D, Mst77F and protamine deposition. A functional approach based on analysis of mutants of histone-modifying enzymes is difficult, as all characterised histone-modifying enzymes are already active during Drosophila development or at least in spermatogonia and spermatocytes. Therefore a tissue-specific knock-out mutant would most probably exhibit arrest of spermatogenesis before meiosis, rendering it useless for experimental purposes (Rathke, 2007).
At the first glance, it might seem surprising that histones and all their modifications are removed. Instead of specifically reverting the differentially modified histones to their unmodified state, they are removed together with all histones. This might allow the paternal genome to form nucleosomes with unmodified histones after fertilisation and before zygote formation. Thus, the paternal genome starts embryogenesis with a nucleosomal chromatin lacking histone modifications (Rathke, 2007).
The data show that most of the histones are removed between the early and late canoe stage; such a process requires a loosening of contact between the histones and DNA, which in turn requires an unwinding of the chromatin structure. It is proposed that this unwinding process is facilitated by DNA nicks as they were widespread at this stage of chromatin reorganisation. Finally, Tpl94D, UbcD6 and SUMO were also observed to accumulate in the chromatin during this process. DNA breaks, Tpl94D, UbcD6 and SUMO were no longer detectable when protamines were fully expressed. Thus, it is proposed that all these proteins and the DNA breaks act together in an unknown manner to allow chromatin remodelling (Rathke, 2007).
The CTCF protein is present during pre-meiotic stages in the nucleus and stays associated with the chromosomes during meiosis. After meiosis, however, strong localisation to the nucleus is detected during the transition from round spermatid nuclei to the early canoe stage of spermiogenesis. It is speculated that CTCF might set borders in the chromatin for the histone modifications, which are characteristic of the canoe stage, such as acetylation and ubiquitylation. CTCF is visible for longer than histones and disappears together with active RNA polymerase II. CTCF might maintain chromatin accessibility to RNA polymerase II since a few genes are known to be transcribed at this time. In addition, transient occurrence of RNA polymerase II at the late canoe stage might require CTCF to insulate active genes from inactive ones. This idea needs to be tested in tissue-specific CTCF loss-of-function mutants; such mutants are, however, currently unavailable (Rathke, 2007).
The question of whether histone removal is dependent on a signal that monitors the start of protamine and Mst77F mRNA translation was addressed. Both histone modification and degradation are indistinguishable from the wild-type in loss-of-function mutants of Mst35Ba and Mst35Bb, the genes encoding protamine A and B, respectively. Also in nc3 mutants of Mst77F, histone removal is not disturbed. It is concluded that N-terminal tail modification of histones and histone degradation, on the one hand, and protamine deposition, on the other, are controlled by different pathways in the cell (Rathke, 2007).
In mammals, it is well known that after meiosis the nucleosomal conformation is lost. This is accompanied by the appearance of testis-specific linker histones. So far, no linker histone variants have been identified in Drosophila, but variants of H2A (H2AvD) and H3 (H3.3) are known. In mammals, histones are hyper-acetylated before being displaced from the DNA, and phosphorylation and ubiquitylation have also been proposed to occur. For Drosophila, H2A mono-ubiquitylation and a strong increase in H4 acetylation occur shortly before histone removal and degradation. In mammals, histones are replaced first by transition proteins (major types: TP1 and TP2). This study identified the high mobility group protein Tpl94D, a first probable candidate for a functional homologue of mammalian transition proteins. In mammals, transition proteins are subsequently replaced by protamines leading to chromatin with a doughnut structure. In Drosophila, it has recently been shown that the sperm nucleus also contains protamines. Protamines A and B are encoded by two closely related protamine genes, Mst35Ba and Mst35Bb. In addition, the identification of Mst77F shows that sperm nuclei contain at least one further abundant chromatin component. Moreover, in human sperm several new putative protamines have been identified by 2D gel electrophoresis and protein sequencing. In mammals, this chromatin reorganisation is essential for male fertility. Male flies carrying the deletion protDelta38.1, where both protamines as well as three additional ORFs are removed, show severely reduced fertility (Rathke, 2007).
In summary, a step-by-step scheme is proposed for chromatin reorganisation: (1) histone modifications lead to subsequent histone removal and degradation; (2) the exposed chromatin becomes nicked, resulting in DNA breaks; (3) Tpl94D deposition constitutes an intermediate stage that triggers subsequent protamine-based chromatin organisation (Rathke, 2007).
Since many features concerning spermiogenesis are conserved between Drosophila and mammals, it is proposed that Drosophila is an ideal system to gain further insight into the mechanism of chromatin reorganisation in spermatid nuclei, a process that is crucial for male fertility (Rathke, 2007).
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The vast majority of the genome is transcriptionally silent in post-meiotic stages. This is accompanied by multiple histone modifications that persist from pre-meiotic stages and indicate silencing such as H3K9 and H3K27 methylation. These modifications do not change significantly during post-meiotic stages, which is in agreement with the hypothesis that these modifications predominantly play a role in maintaining transcriptional silencing. Previously, phosphorylation of histones have been analysed during spermatogenesis. Phosphorylated histone H4S1 and H3S10 are present during meiotic divisions. H3S10 phosphorylation is hardly detectable after meiosis, whereas phosphorylation of H4S1 persists until chromatin compaction starts.
The primary spermatocyte phase is characterised by a high level of transcriptional activity of housekeeping genes. In addition, genes are transcribed that are needed for the subsequent steps in spermatogenesis, as the majority of transcription ceases once meiotic division starts. H4 acetylation and H3K4 and H4R3 methylation of histones were investigated. These histone modifications, which are indicative of transcriptional activity, persist until histone degradation.
It might be that H4 hyper-acetylation, as postulated for mammals and/or other secondary modifications of histones are the first step towards histone removal. The fact that these modifications are conserved between mammals and flies adds support to this hypothesis. Indeed, histone H4 acetylation is very pronounced at the canoe stage and de novo mono-ubiquitylation of histone H2A is seen in round spermatids. Both types of histone modifications are proposed to be necessary for opening the chromatin and decreasing the contact between DNA and histones. The fact that histone H2A mono-ubiquitylation vanishes before the early canoe stage, thus before the hyper-acetylation of histone H4, leads to thinking about a stepwise remodelling of the chromatin. This study proposes that these histone modifications open the chromatin, so that enzymes and regulators have access to histone-based chromatin and can induce and prepare the reorganisation of the genome in the male germline.
Genes Involved in spermatogenesis
Genes involved in organ development
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