Germline Stem Cell Division Research Articles

Direct Control of Germline Stem Cell Division and Cyst Growth by Neural Insulin in Drosophila

Stem cells reside in specialized niches that provide signals required for their maintenance and division. Tissue-extrinsic signals can also modify stem cell activity, although this is poorly understood. Here, we report that neural-derived Drosophila insulin-like peptides (DILPs) directly regulate germline stem cell division rate, demonstrating that signals mediating the ovarian response to nutritional input can modify stem cell activity in a niche-independent manner. We also reveal a crucial direct role of DILPs in controlling germline cyst growth and vitellogenesis.

Structural polarity and dynamics of male germline stem cells in the milkweed bug (Oncopeltus fasciatus).

The male germline stem cells (GSCs) of the milkweed bug present an extraordinary structural polarity that is, to our knowledge, unequalled by any other type of stem cells. They consist of a perikaryon and numerous projections arising from the cell pole directed toward the apical cells, the proposed niche of the GSCs. The projections can traverse a considerable distance until their terminals touch the apical cells. From hatching until death, the GSC projections undergo conspicuous changes, the sequence of which has been deduced from observations of all developmental stages. Projection formation starts from lobular cell protrusions showing trabecular ingrowths of the cell membrane. Finger-like projections result from a process of growth and "carving out". The newly formed projections contain mostly only free ribosomes other than a few mitochondria. A stereotyped degradation process commences in the projection terminals: profiles of circular, often concentric, cisternae of rough endoplasmic reticulum appear and turn into myelin bodies, whereas mitochondria become more numerous. The cytoplasm vesiculates, lysosomal bodies appear, and mitochondria become swollen. At the same time, the projection terminals are segregated by transverse ingrowths of the cell membrane. Finally, autophagic vacuoles and myelin bodies fill the segregated terminals, which then rupture. Simultaneously, new projections seem to sprout from the perikaryon of the GSCs. These dynamics, which are not synchronized among the GSCs, indicate that a novel type of signal exchange and transduction between the stem cells and their niche is involved in the regulation of asymmetric versus symmetric division of GSCs.

A discrete transcriptional silencer in the bam gene determines asymmetric division of the Drosophila germline stem cell.

The Drosophila germline lineage depends on a complex microenvironment of extrinsic and intrinsic factors that regulate the self-renewing and asymmetric divisions of dedicated stem cells. Germline stem cells (GSCs) must express components of the Dpp cassette and the translational repressors Nanos and Pumilio, whereas cystoblasts require the bam and bgcn genes. Bam is especially attractive as a target of GSC differentiation factors because current evidence indicates that bam is both necessary and sufficient for cystoblast differentiation. In this paper, we have sought to distinguish between mutually exclusive transcriptional or post-transcriptional mechanisms as the primary regulators of bam expression in GSCs and cystoblasts. We find that bam transcription is active in young germ cells but is repressed specifically in GSCs. Activation depends on a 50 bp fragment that carries at least one germ cell-specific enhancer element. A non-overlapping 18 bp sequence carries a transcriptional silencer that prevents bam expression in the GSC. Promoters lacking this silencer cause bam expression in the GSC and concomitant GSC loss. Thus, asymmetry of the GSC division can be reduced to identifying the mechanism that selectively activates the silencer element in GSCs.

Mutations of stonewall disrupt the maintenance of female germline stem cells in Drosophila melanogaster.

Germline stem cells located at the anterior tip of the adult Drosophila melanogaster ovary are critical to the continuous production of mature eggs. Following germline stem cell division, one daughter cell remains a stem cell, while the other becomes a cystoblast committed to differentiation. In this study it was shown that mutations in the putative transcription factor stonewall (stwl) disrupted the maintenance of female germline stem cells. The stwl mutations resulted in a loss of germline stem cells, causing a rapid decrease in egg chamber production. The egg chambers developed only to a limited extent before degenerating. The four mitotic cystocyte divisions were frequently inhibited by stwl mutations. Furthermore, some stwl germaria from newly emerged females completely lacked both stem cells and developing cysts and had a strong reduction in size. The argument is presented here that stwl is involved in the continuation of cell division during female germline development.

Yb Modulates the Divisions of Both Germline and Somatic Stem Cells through piwi- and hh-Mediated Mechanisms in the Drosophila Ovary

The coordinated division of distinctive types of stem cells within an organ is crucial for organogenesis and homeostasis. Here we show genetic interactions among fs(1)Yb ( Yb), piwi, and hedgehog ( hh) that regulate the division of both germline stem cells (GSCs) and somatic stem cells (SSCs), the two constituent stem cell populations of the Drosophila ovary. Yb is required for both GSC and SSC divisions; loss of Yb function eliminates GSCs and reduces SSC division, while Yb overexpression increases GSC number and causes SSC overproliferation. We also show that Yb acts via the piwi- and hh-mediated signaling pathways that emanate from the same signaling cells to control GSC and SSC division, respectively. hh signaling also has a minor effect in GSC division.

Asymmetric germ cell division and oocyte determination during Drosophila oogenesis

Early oogenesis in Drosophila, with a stereotypic pattern of germ cell division and differentiation, provides an attractive model for studying cell lineage and patterning. Drosophila oogenesis is initiated when a germline stem cell divides asymmetrically to produce a daughter stem cell and a cytoblast. The cystoblast then undergoes four rounds of incomplete mitoses to form a 16-cell cyst, accompanied by the formation of the fusome. Within the cyst, one of the two cells with four intercellular bridges differentiates into an oocyte while the rest become nurse cells. The oocyte then translocates within the cyst to a posterior position, which defines the anterio-posterior axis of the future embryo. Recent studies have shown that the asymmetric germline stem cell division is controlled by somatic signaling involving piwi, fs(1)Yb, and the dpp pathway as well as by intrinsic mechanisms involving pumilio, nanos, arrest, bag-of-marbles, and the spectrosome-the fusome precursor in the stem cells and the cystoblast. The spectrosome in the cystoblast appears to play an important role in polarized fusome growth during cyst formation. The fusome may guide the formation of a polarized microtubule network for the intracyst transport of certain RNAs and proteins to the cystocyte destined to become the oocyte. Genes such as egalitarian, Bicaudal D, stonewall, and encore are important for oocyte determination, while differential adhesion between the oocyte and its surrounding prefollicle cells, as mediated by armadillo, alpha-catenin, shotgun, and the spindle genes, is crucial for oocyte translocation. Early oogenesis shares many parallel features to early spermatogenesis, although distinct differences are also observed at both the phenomenological and mechanistic levels. The study of oogenesis, progressing at an exciting rate, contributes significantly to our understanding of the mechanisms underlying proliferation, differentiation, and patterning.

Piwi encodes a nucleoplasmic factor whose activity modulates the number and division rate of germline stem cells.

piwi represents the first class of genes known to be required for stem cell self-renewal in diverse organisms. In the Drosophila ovary, piwi is required in somatic signaling cells to maintain germline stem cells. Here we show that piwi encodes a novel nucleoplasmic protein present in both somatic and germline cells, with the highly conserved C-terminal region essential for its function. Removing PIWI protein from single germline stem cells significantly decreases the rate of their division. This suggests that PIWI has a second role as a cell-autonomous promoter of germline stem cell division. Consistent with its dual function, over-expression of piwi in somatic cells causes an increase both in the number of germline stem cells and the rate of their division. Thus, PIWI is a key regulator of stem cell division - its somatic expression modulates the number of germline stem cells and the rate of their division, while its germline expression also contributes to promoting stem cell division in a cell-autonomous manner.

The Drosophila pumilio gene encodes two functional protein isoforms that play multiple roles in germline development, gonadogenesis, oogenesis and embryogenesis.

The pumilio (pum) gene plays an essential role in embryonic patterning and germline stem cell (GSC) maintenance during oogenesis in Drosophila. Here we report on a phenotypic analysis using pum(ovarette) mutations, which reveals multiple functions of pum in primordial germ cell proliferation, larval ovary formation, GSC division, and subsequent oogenic processes, as well as in oviposition. Specifically, by inducing pum(-) GSC clones at the onset of oogenesis, we show that pum is directly involved in GSC division, a function that is distinct from its requirement in primordial germ cells. Furthermore, we show that pum encodes 156- and 130-kD proteins, both of which are functional isoforms. Among pum(ovarette) mutations, pum(1688) specifically eliminates the 156-kD isoform but not the 130-kD isoform, while pum(2003) and pum(4277) specifically affect the 130-kD isoform but not the 156-kD isoform. Normal doses of both isoforms are required for the zygotic function of pum, yet either isoform alone at a normal dose is sufficient for the maternal effect function of pum. A pum cDNA transgene that contains the known open reading frame encodes only the 156-kD isoform and rescues the phenotype of both pum(1688) and pum(2003) mutants. These observations suggest that the 156- and 130-kD isoforms can compensate for each other's function in a dosage-dependent manner. Finally, we present molecular evidence suggesting that the two PUM isoforms share some of their primary structures.

The self-renewing mechanism of stem cells in the germline

Germline stem cells (GSCs) are a self-renewing population of germ cells that serve as the source of gametes in diverse organisms. Current research suggests that the self-renewing division of GSCs is controlled both by somatic signaling and by intracellular mechanisms such as differential gene expression, asymmetric cytoskeletal organization, and the cell cycle machinery. These findings provide a framework for the further study of GSCs and stem cell renewal in general.

Decapentaplegic Is Essential for the Maintenance and Division of Germline Stem Cells in the Drosophila Ovary

Stem cells are thought to occupy special local environments, or niches, established by neighboring cells that give them the capability for self-renewal. Each ovariole in the Drosophila ovary contains two germline stem cells surrounded by a group of differentiated somatic cells that express hedgehog and wingless. Here we show that the BMP2/4 homolog decapentaplegic (dpp) is specifically required to maintain female germline stem cells and promote their division. Overexpression of dpp blocks germline stem cell differentiation. Conversely, mutations in dpp or its receptor ( saxophone) accelerate stem cell loss and retard stem cell division. We constructed mutant germline stem cell clones to show that the dpp signal is directly received by germline stem cells. Thus, dpp signaling helps define a niche that controls germline stem cell proliferation.

The tao of stem cells in the germline.

Germline stem cells (GSCs) are the self-renewing population of germ cells that serve as the source for gametogenesis. GSCs exist in diverse forms, from those that undergo strict self-renewing asymmetric divisions in Drosophila to those that maintain their population by balancing between mitosis and differentiation in Caenorhabditis elegans. Most vertebrate spermatogonial GSCs appear to adopt an intermediate strategy. In most animals, GSCs are established during preadult gonadogenesis following the proliferation and migration of embryonic primordial germ cells. GSCs produce numerous gametes throughout the sexually active period of adult life. The establishment and self-renewing division of GSCs are controlled by extracellular signals such as hormones from the hypothalamic-pituitary axis and local interactions between GSCs and their neighboring cells. These extracellular signals may then influence differential gene expression, cell cycle machinery, and cytoskeletal organization of GSCs for their formation and/or divisional asymmetry. In addition, the GSC mechanism is related to that for germline and sex determination. Current knowledge has provided a solid framework for further study of GSCs and stem cells in general.

Spectrosomes and Fusomes Anchor Mitotic Spindles during Asymmetric Germ Cell Divisions and Facilitate the Formation of a Polarized Microtubule Array for Oocyte Specification inDrosophila

In theDrosophilaovary, membrane skeletal proteins such as the adducin-like Hts protein(s), spectrin, and ankyrin are found in the spectrosome, an organelle in germline stem cells (GSC) and their differentiated daughter cells (cystoblasts). These proteins are also components of the fusome, a cytoplasmic structure that spans the cystoblast's progeny that develop to form a germline cyst consisting of 15 nurse cells and an oocyte. Spectrosomes and fusomes are associated with one pole of spindles during mitosis and are implicated in cyst formation and oocyte differentiation. Here we show that the asymmetric behavior of the spectrosome persists throughout the cell cycle of GSC. Eliminating the spectrosome by thehtslmutation leads to randomized spindle orientation, suggesting that the spectrosome anchors the spindle to ensure the asymmetry of GSC division; eliminating the fusome in developing cysts results in defective spindles and randomized spindle orientation as well as asynchronous and reduced cystocyte divisions. These observations suggest that fusomes are required for the proper formation and asymmetric orientation of mitotic spindles. Moreover, they reinforce the notion that fusomes are required for the four synchronous divisions of the cystoblast leading to cyst formation. Inhtslcysts which lack fusomes and fail to incorporate ahtsgene product(s) into ring canals following cyst formation, polarized microtubule networks do not form, the dynamics of cytoplasmic dynein is disrupted, andoskarandorbRNAs fail to be transported to the future oocyte. These observations support the proposed role of fusomes and ring canals in organizing a polarized microtubule-based transport system for RNA localization that leads to oocyte differentiation.

A novel group of pumilio mutations affects the asymmetric division of germline stem cells in the Drosophila ovary.

Germline stem cells play a pivotal role in gametogenesis; yet little is known about how they are formed, how they divide to self-renew, and how these processes are genetically controlled. Here we describe the self-renewing asymmetric division of germline stem cells in the Drosophila ovarian germline, as marked by the spectrosome, a cytoplasmic structure rich in membrane skeletal proteins. The ontogeny of the spectrosome marks the lineage of germline stem cells. We identified two new groups of mutations in which the divisional asymmetry is disrupted. The first, which we refer to as ovarette (ovt) mutations, was shown to correspond to a novel class of mutations in the pumilio locus. Since pumilio is known to posttranscriptionally repress the expression of target genes at earlier stages of germ cell development, our results suggest that a similar activity is needed to maintain germ line stem cells. We have also identified a second and novel gene, piwi, whose mutations abolish germline stem cell division.

Germline Stem Cell Division and Egg Chamber Development in Transplanted Drosophila Germaria

Germline and somatic stem cells reside within the anterior region (or "germarium") of each ovariole in the Drosophila ovary. When individual germaria were dissected free of developing eggs and sheath tissue and transplanted into the abdominal cavity of a host fly, they regenerated ovariole-like structures and continuously supported the entire process of oogenesis, indicating that the stem cells remained functional. This system allowed us to measure the duration of several stages in oogenesis and to analyze the role of specific germarial cells in providing stem cell function. Laser ablation of presumptive germline stem cells near the apical tip prior to transplantation blocked the production of new germline cysts, but allowed previously initiated cysts to complete development. This confirmed the location of germline stem cells and showed that subsequent development of preexisting cysts did not require continued cyst production. Ablation of a distinct group of somatic cells lying close to the germline stem cells ("the terminal filament") increased the rate of oogenesis by approximately 40%, suggesting that the terminal filament may negatively regulate stem cell division.