A Temporal Signature of Epidermal Growth Factor Signaling Regulates the Differentiation of Germline Cells in Testes of Drosophila melanogaster

Tissue replenishment from stem cells follows a precise cascade of events, during which stem cell daughters first proliferate by mitotic transit amplifying divisions and then enter terminal differentiation. Here we address how stem cell daughters are guided through the early steps of development. In Drosophila testes, somatic cyst cells enclose the proliferating and differentiating germline cells and the units of germline and surrounding cyst cells are commonly referred to as cysts. By characterizing flies with reduced or increased Epidermal Growth Factor (EGF) signaling we show that EGF triggers different responses in the cysts dependent on its dose. In addition to the previously reported requirement for EGF signaling in cyst formation, a low dose of EGF signaling is required for the progression of the germline cells through transit amplifying divisions, and a high dose of EGF signaling promotes terminal differentiation. Terminal differentiation was promoted in testes expressing a constitutively active EGF Receptor (EGFR) and in testes expressing both a secreted EGF and the EGFR in the cyst cells, but not in testes expressing either only EGF or only EGFR. We propose that as the cysts develop, a temporal signature of EGF signaling is created by the coordinated increase of both the production of active ligands by the germline cells and the amount of available receptor molecules on the cyst cells.     

Ultrastructural study of spermatogenesis in Oscarella lobularis (Porifera,Demospongiae)

Summary

The various phases of spermatogenesis in the demosponge Oscarella lobularis were studied by electron microscopy. Spermatogenesis occurs within spermatic cysts, which are presumed to derive from choanocyte chambers by transformation of choanocytes into spermatogonia. Germ cells develop asynchronously within spermatocysts, and cytoplasmic bridges, indicating incomplete cells division, connect several germ cells. Attached spermatogonia suggest gonial generations. Spermatocytes I typically show the presence of synaptonemal complexes indicating meiotic divisions. Spermatocytes II have a small size probably because of the meiotic divisions of spermatocytes I. Spermatids are characterized by an acrosome, a big mitochondrion and a peripheral sheath of condensed chromatin surrounding a clearer central area in the nucleus. The mature spermatozoon shows a lateral flagellum and a flattened acrosome capping the nucleus. The phylogenetic implications of some features of the spermatozoon are suggested.     

Evolution and development of polarized germ cell cysts: new insights from a polychaete worm, Ophryotrocha labronica

Polarized oogenic cysts are clonal syncytia of germ cells in which some of the sister cells (cystocytes) differentiate not as oocytes, but instead as nurse cells: polyploid cells that support oocyte development. The intricate machinery required to establish and maintain divergent cell fates within a syncytium, and the importance of associated oocyte patterning for subsequent embryonic development, have made polarized cysts valuable subjects of study in developmental and cell biology. Nurse cell/oocyte specification is best understood in insects, particularly Drosophila melanogaster. However, polarized cysts have evolved independently in several other animal phyla. We describe the differentiation of female cystocytes in an annelid worm, the polychaete Ophryotrocha labronica. These worms are remarkable for their elegantly simple cysts, which comprise a single oocyte and nurse cell, making them an appealing complement to insects as subjects of study. To elucidate the process of cystocyte differentiation in O. labronica, we have constructed digital 3D models from electron micrographs of serially sectioned ovarian tissue. These models show that 2-cell cysts arise by fragmentation of larger “parental” cysts, rather than as independent units. The parental cysts vary in size and organization, are produced by asynchronous, indeterminate mitotic divisions of progenitor cystoblasts, and lack fusome-like organizing organelles. All of these characteristics represent key cytological differences from “typical” cyst development in insects like D. melanogaster. In light of such differences and the plasticity of female cyst structure among other animals, we suggest that it is time to reassess common views on the conservation of oogenic cysts and the importance of cysts in animal oogenesis generally.     

Cytology of lepidoptera. III. Giant cysts: A morphological trait of apyrene spermatogenesis in an Ephestia kuehniella strain

A comparative investigation of testicular eupyrene cysts (in larvae) and apyrene cysts (in pupae) of Ephestia kuehniella laboratory strains was conducted using light and electron microscopy. Eupyrene cysts in the first meiotic division contained 64 spermatocytes, which showed only moderate asynchrony. In one of the strains, a wild-type strain, L, normal-sized cysts occurred together with abnormally large cysts. These are called giant cysts in this article. One of the premeiotic cysts, early giant cysts, studied in detail, contained approximately a fourfold number of cells compared with the number in a eupyrene cyst of the same stage. In cysts harboring spermatocytes and spermatids, late giant cysts, cell differentiation was highly asynchronous. Failure in one of two control mechanisms in early cyst development may have caused the appearance of the cysts. Control of cell division might have been sloppy in apyrene spermatogonia. Hence, the spermatogonia within the cyst could have passed through additional division cycles. Alternatively, the giant cysts may have originated from more than one predefinitive gonial cell enclosed in a common envelope of sheath cells. As a third possibility, giant cysts could have arisen by fusion of normal cysts at a later stage. In either case, this is evidence that separation of eupyrene and apyrene pathways is earlier than was previously expected. In two other Ephestia strains, apyrene sperm development proceeded without formation of giant cysts. One was a mutant strain, a, and the other one was a recently established wild-type strain, Sbr. Apyrene sperm development is considered an example of degenerate evolution in which enhanced variability between species and even between populations of one species is a common phenomenon.     

Chromosome pairing in the oögonial cells of Drosophila melanogaster

Pairing between two nonhomologous chromosomes, one a free X-duplication and the other a free fourth chromosome, has been observed cytologically with high frequencies in the oögonial cells of Drosophila melanogaster. The frequencies of nonhomologous pairing ranged from 27 to 47% and showed a positive correlation with the similarity in size between the two participating nonhomologues. Partial homology increased pairing frequency between nonhomologues in the oögonial cell, in contrast to the behavior of the same nonhomologues at distributive pairing in the oöcyte, where pairing is strictly size-dependent. Pairing between homologues in the same oögonial cells occurred at a frequency of only 71% and was higher for the autosomes (73%) than for the sex chromosomes (66%). An increased frequency of homologous pairing was found for older gonial cysts (4-cell, 72.0% ; 8-cell, 76.1%) as compared with younger cysts (1-cell, 59.1% ; 2-cell, 53.1%).Research sponsored by the U.S. Atomic Energy Commission under contract with the Union Carbide Corporation.     

Cell proliferation and differentiation kinetics during spermatogenesis inHydra carnea

Summary Spermatogenesis inHydra carnea was investigated. The cell proliferation and differentiation kinetics of intermediates in the spermatogenesis pathway were determined, using quantitative determinations of cell abundance, pulse and continuous labelling with³H-thymidine and nuclear DNA measurements. Testes develop in the ectoderm of male hydra as a result of interstitial cell proliferation. Gonial stem cells and proliferating spermatogonia have cell cycles of 28 h and 22 h, respectively. Stem cells undergo four, five or six cell divisions prior to meiosis which includes a premeiotic S+G2 phase of 20 h followed by a long meiotic prophase (22 h).Spermatid differentiation requires 12–29 h. When they first appear, testes contain only proliferating spermatogonia; meiotic and postmeiotic cells appear after 2 and 3 days, respectively and release of mature sperm begins after 4 days. Mature testes produce about 27,000 sperm per day over a period of 4–6 days: about 220 gonial stem cells per testis are required to support this level of sperm differentiation. Further results indicate that somatic (e.g. nematocyte) differentiation does not occur in testes although it continues normally in ectodermal tissue outside testes. Our results support the hypothesis that spermatogenesis is controlled locally in regions of the ectoderm where testes develop.     

Meiosis in DROSOPHILA MELANOGASTER. III. the Effect of Orientation Disruptor (ord) on Gonial Mitotic and the Meiotic Divisions in Males

Orientation disruptor (ord), a meiotic mutant that is recombination defective in females and disjunction defective in males and females, has been analyzed using serial section electron and light microscopy. From analysis of primary spermatocytes we have confirmed that ord males are defective in some aspect of the mechanism(s) that holds sister chromatids together during meiosis. In addition, we have determined that ord causes high frequencies of nondisjunction during spermatogonial mitotic divisions, as well as during the meiotic divisions. Mitotic nondisjunction involves the large autosomes more frequently than the sex chromosomes or chromosome 4 and results in high frequencies of primary spermatocytes that are either monosomic or trisomic for chromosome 2 or 3. Abnormalities in spermatocyte cyst formation are also observed in males homozygous for ord. These abnormalities include loss of regulation of meiotic synchrony and the number of gonial cell divisions.     

Protease nexins: cell-secreted proteins that mediate the binding, internalization, and degradation of regulatory serine proteases

Cortisol and insulin stimulated exponential growth of normal human mammary epithelium in short-term monolayer culture. The response of cells depended on their organization into "growth units" on the surface of the culture dish; single cells did not respond. Growth of cells in the units ceased after only 3-4 doublings, ending in terminal differentiation. The 3-4 divisions that occurred in response to insulin and cortisol and that resulted in terminal differentiation, were not inhibited by short-range signals normally transmitted at population confluence. When growing above confluence density in response to hormones, cells reduced volume to accommodate the "terminal differentiation" divisions while still largely preserving a monolayer. The longevity of populations of normal cells (9-14 divisions), which occurred in 3 or 4 passages, exceeded the average longevity of individual cells in one passage (3-4 divisions). This disparity between real and apparent longevity was due to the inhibition of growth of divisional cells within growth units, which occurred in concert with terminal differentiation of other cells in these units. Inhibited cells could be recruited to undergo terminal differentiation divisions in response to cortisol and insulin, but only when the growth units were disrupted and terminally differentiated cells were eliminated, which occurred at subculture. We refer to the inhibition of growth that occurs in growth units as "renewal inhibition" to distinguish it from population-wide "confluence inhibition" and to emphasize three other aspects: (1) it occurred in terminally differentiating growth units; (2) it occurred in the continued presence of an inductive hormonal stimulus for differentiative growth; and (3) it conserved less differentiated cells for recruitment to terminal differentiation. There are parallels between renewal inhibition in vitro and the signals that restrain growth of mammary cells in 'growth buds" in vivo to preserve their capacity for multiple cycles of secretory differentiation. Differences in the behavior of normal and malignant breast cells in vitro suggest that renewal inhibition, rather than confluence inhibition, may be an important locus of growth control alteration in malignant transformation.     

The abnormal spindle protein is required for germ cell mitosis and oocyte differentiation during Drosophila oogenesis

In this study, we present evidence that the asp function is required in oogenesis for germline cell divisions as well as for cyst polarity and oocyte differentiation. Consistent with previously described roles in spindle organization during Drosophila meiosis and mitosis, asp mutation leads to severe defects in spindle microtubule organization within the germarium. The mitotic spindles of the mutant cystocytes are composed by wavy microtubules and have abnormal poles that often lack gamma-tubulin. The fusome structure is also compromised. In the absence of asp function, the cystocyte divisions fail resulting in egg chamber with fewer than 16 germ cells. Moreover, the microtubule network within the developing germline cysts may assemble incorrectly in turn affecting the microtubule based transport of the specific determinants that is required during mid-oogenesis for the oocyte differentiation program.     

Developmental Coordination of Gamete Differentiation with Programmed Cell Death in Sporulating Yeast

The gametogenesis program of the budding yeast Saccharomyces cerevisiae, also known as sporulation, employs unusual internal meiotic divisions, after which all four meiotic products differentiate within the parental cell. We showed previously that sporulation is typically accompanied by the destruction of discarded immature meiotic products through their exposure to proteases released from the mother cell vacuole, which undergoes an apparent programmed rupture. Here we demonstrate that vacuolar rupture contributes to de facto programmed cell death (PCD) of the meiotic mother cell itself. Meiotic mother cell PCD is accompanied by an accumulation of depolarized mitochondria, organelle swelling, altered plasma membrane characteristics, and cytoplasmic clearance. To ensure that the gametes survive the destructive consequences of developing within a cell that is executing PCD, we hypothesized that PCD is restrained from occurring until spores have attained a threshold degree of differentiation. Consistent with this hypothesis, gene deletions that perturb all but the most terminal postmeiotic spore developmental stages are associated with altered PCD. In these mutants, meiotic mother cells exhibit a delay in vacuolar rupture and then appear to undergo an alternative form of PCD associated with catastrophic consequences for the underdeveloped spores. Our findings reveal yeast sporulation as a context of bona fide PCD that is developmentally coordinated with gamete differentiation.     

Modification of radiation-induced genetic damage in Drosophila melanogaster male germ cells with nucleic acid precursors. 3. Effects on the premeiotic cells

Using a 2-day brood pattern, the effect of 5-bromodeoxyuridine (BUdR) or 5-bromodeoxycytidine (BCdR) pre-treatment on the radiation-induced yield of sex-linked recessive lethals and translocations was studied in the spermatocytes and late gonial cells (p.i. DNA synthesis cells) of D. melanogaster. The p.i. DNA synthesis cells were irradiated (I.2 kR γ-radiation) in the pre-meiotic or post-meiotic stage. Irradiation of p.i. DNA synthesis cells in the pre-meiotic stage resulted in enhanced lethal frequency with BUdR (3.0%) and BCdR (2.9%) over the other pre-treatment conditions: saline (S), thymidine (TdR) and deoxycitydine (CdR) in the spermatocytes but not in the late gonial cells. The radiosensitizing property was evident with BCdR even when the p.i. DNA synthesis cells were irradiated in the post-meiotic stage; but not with BUdR pre-treatment. Probable reasons for the contradicting results reported in the literature were discussed.     

Evidence for asynchronous mitotic cell divisions in secondary spermatogonia of Drosophila

Examination of germ cell numbers within premeiotic as well as postmeiotic cysts of various Drosophila species gave evidence against any strict synchrony of mitotic cell division in secondary spermatogonia. The evidence was based on numbers of germ cells in primary spermatocyte cysts and spermatid bundles. Each species examined had its own distribution of primary spermatocyte cyst types in pupal testes, and the most common cyst type did not necessarily contain 2, 4, 8, 16 or (2) ⁿ germ cells which implies asynchrony of the previous spermatogonial divisions. Similar but not exactly the same distributions of germ cells were found in adult spermatid bundles, if allowances were made for a 4-fold increase in germ cell number during meiosis. This observation gives support to the operation of an age-dependent factor which controls germ cell numbers within cysts [1], The data thus suggest that the commonly accepted concept of a (2) ⁿ increase of spermatogonia via synchronous mitotic divisions is not true for the species of Drosophila studied.     

The fusome mediates intercellular endoplasmic reticulum connectivity in Drosophila ovarian cysts

Drosophila ovarian cysts arise through a series of four synchronous incomplete mitotic divisions. After each round of mitosis, a membranous organelle, the fusome, grows along the cleavage furrow and the remnants of the mitotic spindle to connect all cystocytes in a cyst. The fusome is essential for the pattern and synchrony of the mitotic cyst divisions as well as oocyte differentiation. Using live cell imaging, green fluorescent protein-tagged proteins, and photobleaching techniques, we demonstrate that fusomal endomembranes are part of a single continuous endoplasmic reticulum (ER) that is shared by all cystocytes in dividing ovarian cysts. Membrane and lumenal proteins of the common ER freely and rapidly diffuse between cystocytes. The fusomal ER mediates intercellular ER connectivity by linking the cytoplasmic ER membranes of all cystocytes within a cyst. Before entry into meiosis and onset of oocyte differentiation (between region 1 and region 2A), ER continuity between cystocytes is lost. Furthermore, analyses of hts and Dhc64c mutants indicate that intercellular ER continuity within dividing ovarian cysts requires the fusome cytoskeletal component and suggest a possible role for the common ER in synchronizing mitotic cyst divisions.     

Post-embryonic cell lineages of the nematode,Caenorhabditis elegans

The number of nongonadal nuclei in the free-living soil nematode Caenorhabditis elegans increases from about 550 in the newly hatched larva to about 810 in the mature hermaphrodite and to about 970 in the mature male. The pattern of cell divisions which leads to this increase is essentially invariant among individuals; rigidly determined cell lineages generate a fixed number of progeny cells of strictly specified fates. These lineages range in length from one to eight sequential divisions and lead to significant developmental changes in the neuronal, muscular, hypodermal, and digestive systems. Frequently, several blast cells follow the same asymmetric program of divisions; lineally equivalent progeny of such cells generally differentiate into functionally equivalent cells. We have determined these cell lineages by direct observation of the divisions, migrations, and deaths of individual cells in living nematodes. Many of the cell lineages are involved in sexual maturation. At hatching, the hermaphrodite and male are almost identical morphologically; by the adult stage, gross anatomical differences are obvious. Some of these sexual differences arise from blast cells whose division patterns are initially identical in the male and in the hermaphrodite but later diverge. In the hermaphrodite, these cells produce structures used in egg-laying and mating, whereas, in the male, they produce morphologically different structures which function before and during copulation. In addition, development of the male involves a number of lineages derived from cells which do not divide in the hermaphrodite. Similar postembryonic developmental events occur in other nematode species.     

Localization and function of Bam protein require the benign gonial cell neoplasm gene product.

Division of a female Drosophila stem cell produces a daughter stem cell and a cystoblast. The cystoblast produces a syncytial cluster of 16 cells by precisely four mitotic divisions and incomplete cytokinesis. Mutations in genes required for cystoblast differentiation, such as bag-of-marbles, block syncytial cluster formation and produce a distinctive "tumorous" or hyperplastic germ cell phenotype. In this paper, we compare the oogenic phenotype of benign gonial cell neoplasm mutations to that of mutations in bam. The data indicate that, like bam, bgcn is required for cystoblast development and that germ cells lacking bgcn become trapped in a stem cell-like state. One indication that germ cells lacking bgcn cannot form cystoblasts is that bgcn stem cells resist genetic ablation by Bam misexpression. Misexpression of Bam eliminates wild-type stem cells, apparently by inducing them to divide as cystoblasts. bgcn stem cells remain active when Bam is misexpressed, probably because they cannot adopt the cystoblast fate. Bgcn activity is not required for Bam protein expression but is essential for the localization of Bam protein to the fusome. Together, the results suggest that Bam and Bgcn cooperatively regulate cystoblast differentiation by controlling localization of Bam protein to the fusome.     

Co-culture with mesenchymal stromal cells increases proliferation and maintenance of haematopoietic progenitor cells

Mesenchymal stromal cells (MSC) have been suggested to provide a suitable cellular environment for in vitro expansion of haematopoietic stem and progenitor cells (HPC) from umbilical cord blood. In this study, we have simultaneously analysed the cell division history and immunophenotypic differentiation of HPC by using cell division tracking with carboxyfluorescein diacetate N -succinimidyl ester (CFSE). Co-culture with MSC greatly enhanced proliferation of human HPC, especially of the more primitive CD34⁺CD38⁻ fraction. Without co-culture CD34 and CD133 expressions decreased after several cell divisions, whereas CD38 expression was up-regulated after some cell divisions and then diminished in fast proliferating cells. Co-culture with MSC maintained a primitive immunophenotype (CD34⁺, CD133⁺ and CD38⁻) for more population doublings, whereas up-regulation of differentiation markers (CD13, CD45 and CD56) in HPC was delayed to higher numbers of cell divisions. Especially MSC of early cell passages maintained CD34 expression in HPC over more cell divisions, whereas MSC of higher passages further enhanced their proliferation rate. Inhibition of mitogen-activated protein kinase 1 (MAPK1) impaired proliferation and differentiation of HPC, but not maintenance of long-term culture initiating cells. siRNA knockdown of N-cadherin and VCAM1 in feeder layer cells increased the fraction of slow dividing HPC, whereas knockdown of integrin beta 1 (ITGB1) and CD44 impaired their differentiation. In conclusion, MSC support proliferation as well as self-renewal of HPC with primitive immunophenotype. The use of early passages of MSC and genetic manipulation of proteins involved in HPC–MSC interaction might further enhance cord blood expansion on MSC.