Ku-Mediated Coupling of DNA Cleavage and Repair during Programmed Genome Rearrangements in the Ciliate Paramecium tetraurelia

During somatic differentiation, physiological DNA double-strand breaks (DSB) can drive programmed genome rearrangements (PGR), during which DSB repair pathways are mobilized to safeguard genome integrity. Because of their unique nuclear dimorphism, ciliates are powerful unicellular eukaryotic models to study the mechanisms involved in PGR. At each sexual cycle, the germline nucleus is transmitted to the progeny, but the somatic nucleus, essential for gene expression, is destroyed and a new somatic nucleus differentiates from a copy of the germline nucleus. In Paramecium tetraurelia, the development of the somatic nucleus involves massive PGR, including the precise elimination of at least 45,000 germline sequences (Internal Eliminated Sequences, IES). IES excision proceeds through a cut-and-close mechanism: a domesticated transposase, PiggyMac, is essential for DNA cleavage, and DSB repair at excision sites involves the Ligase IV, a specific component of the non-homologous end-joining (NHEJ) pathway. At the genome-wide level, a huge number of programmed DSBs must be repaired during this process to allow the assembly of functional somatic chromosomes. To understand how DNA cleavage and DSB repair are coordinated during PGR, we have focused on Ku, the earliest actor of NHEJ-mediated repair. Two Ku70 and three Ku80 paralogs are encoded in the genome of P. tetraurelia: Ku70a and Ku80c are produced during sexual processes and localize specifically in the developing new somatic nucleus. Using RNA interference, we show that the development-specific Ku70/Ku80c heterodimer is essential for the recovery of a functional somatic nucleus. Strikingly, at the molecular level, PiggyMac-dependent DNA cleavage is abolished at IES boundaries in cells depleted for Ku80c, resulting in IES retention in the somatic genome. PiggyMac and Ku70a/Ku80c co-purify as a complex when overproduced in a heterologous system. We conclude that Ku has been integrated in the Paramecium DNA cleavage factory, enabling tight coupling between DSB introduction and repair during PGR.     

Ecdysone signaling opposes epidermal growth factor signaling in regulating cyst differentiation in the male gonad of Drosophila melanogaster

The development of stem cell daughters into the differentiated state normally requires a cascade of proliferation and differentiation steps that are typically regulated by external signals. The germline cells of most animals, in specific, are associated with somatic support cells and depend on them for normal development. In the male gonad of Drosophila melanogaster, germline cells are completely enclosed by cytoplasmic extensions of somatic cyst cells, and these cysts form a functional unit. Signaling from the germline to the cyst cells via the Epidermal Growth Factor Receptor (EGFR) is required for germline enclosure and has been proposed to provide a temporal signature promoting early steps of differentiation. A temperature-sensitive allele of the EGFR ligand Spitz (Spi) provides a powerful tool for probing the function of the EGRF pathway in this context and for identifying other pathways regulating cyst differentiation via genetic interaction studies. Using this tool, we show that signaling via the Ecdysone Receptor (EcR), a known regulator of developmental timing during larval and pupal development, opposes EGF signaling in testes. In spi mutant animals, reducing either Ecdysone synthesis or the expression of Ecdysone signal transducers or targets in the cyst cells resulted in a rescue of cyst formation and cyst differentiation. Despite of this striking effect in the spi mutant background and the expression of EcR signaling components within the cyst cells, activity of the EcR pathway appears to be dispensable in a wildtype background. We propose that EcR signaling modulates the effects of EGFR signaling by promoting an undifferentiated state in early stage cyst cells.     

Daughterless coordinates somatic cell proliferation,differentiation and germline cyst survival during follicle formation in Drosophila

During Drosophila oogenesis two distinct stem cell populations produce either germline cysts or the somatic cells that surround each cyst and separate each formed follicle. From analyzing daughterless (da) loss-of-function, overexpression and genetic interaction phenotypes, we have identified several specific requirements for da(+) in somatic cells during follicle formation. First, da is a critical regulator of somatic cell proliferation. Also, da is required for the complete differentiation of polar and stalk cells, and elevated da levels can even drive the convergence and extension that is characteristic of interfollicular stalks. Finally, da is a genetic regulator of an early checkpoint for germline cyst progression: Loss of da function inhibits normally occurring apoptosis of germline cysts at the region 2a/2b boundary of the germarium, while da overexpression leads to postmitotic cyst degradation. Collectively, these da functions govern the abundance and diversity of somatic cells as they coordinate with germline cysts to form functional follicles.     

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.     

Two Classes of Gap Junction Channels Mediate Soma-Germline Interactions Essential for Germline Proliferation and Gametogenesis in Caenorhabditis elegans

In all animals examined, somatic cells of the gonad control multiple biological processes essential for germline development. Gap junction channels, composed of connexins in vertebrates and innexins in invertebrates, permit direct intercellular communication between cells and frequently form between somatic gonadal cells and germ cells. Gap junctions comprise hexameric hemichannels in apposing cells that dock to form channels for the exchange of small molecules. Here we report essential roles for two classes of gap junction channels, composed of five innexin proteins, in supporting the proliferation of germline stem cells and gametogenesis in the nematode Caenorhabditis elegans. Transmission electron microscopy of freeze-fracture replicas and fluorescence microscopy show that gap junctions between somatic cells and germ cells are more extensive than previously appreciated and are found throughout the gonad. One class of gap junctions, composed of INX-8 and INX-9 in the soma and INX-14 and INX-21 in the germ line, is required for the proliferation and differentiation of germline stem cells. Genetic epistasis experiments establish a role for these gap junction channels in germline proliferation independent of the glp-1/Notch pathway. A second class of gap junctions, composed of somatic INX-8 and INX-9 and germline INX-14 and INX-22, is required for the negative regulation of oocyte meiotic maturation. Rescue of gap junction channel formation in the stem cell niche rescues germline proliferation and uncovers a later channel requirement for embryonic viability. This analysis reveals gap junctions as a central organizing feature of many soma–germline interactions in C. elegans.     

Steroid Signaling within Drosophila Ovarian Epithelial Cells Sex-Specifically Modulates Early Germ Cell Development and Meiotic Entry

Drosophila adult females but not males contain high levels of the steroid hormone ecdysone, however, the roles played by steroid signaling during Drosophila gametogenesis remain poorly understood. Drosophila germ cells in both sexes initially follow a similar pathway. After germline stem cells are established, their daughters form interconnected cysts surrounded by somatic escort (female) or cyst (male) cells and enter meiosis. Subsequently, female cysts acquire a new covering of somatic cells to form follicles. Knocking down expression of the heterodimeric ecdysteroid receptor (EcR/Usp) or the E75 early response gene in escort cells disrupts 16-cell cyst production, meiotic entry and follicle formation. Escort cells lose their squamous morphology and unsheath germ cells. By contrast, disrupting ecdysone signaling in males does not perturb cyst development or ensheathment. Thus, sex-specific steroid signaling is essential for female germ cell development at the time male and female pathways diverge. Our results suggest that steroid signaling plays an important sex-specific role in early germ cell development in Drosophila, a strategy that may be conserved in mammals.     

Germ cells commit somatic stem cells to differentiation following priming by PI3K/Tor activity in the Drosophila testis

How and when potential becomes restricted in differentiating stem cell daughters is poorly understood. While it is thought that signals from the niche are actively required to prevent differentiation, another model proposes that stem cells can reversibly transit between multiple states, some of which are primed, but not committed, to differentiate. In the Drosophila testis, somatic cyst stem cells (CySCs) generate cyst cells, which encapsulate the germline to support its development. We find that CySCs are maintained independently of niche self-renewal signals if activity of the PI3K/Tor pathway is inhibited. Conversely, PI3K/Tor is not sufficient alone to drive differentiation, suggesting that it acts to license cells for differentiation. Indeed, we find that the germline is required for differentiation of CySCs in response to PI3K/Tor elevation, indicating that final commitment to differentiation involves several steps and intercellular communication. We propose that CySC daughter cells are plastic, that their fate depends on the availability of neighbouring germ cells, and that PI3K/Tor acts to induce a primed state for CySC daughters to enable coordinated differentiation with the germline.     

Multiple roles of the F-box protein Slimb in Drosophila egg chamber development

Substrate-specific degradation of proteins by the ubiquitin-proteasome pathway is a precise mechanism that controls the abundance of key cell regulators. SCF complexes are a family of E3 ubiquitin ligases that target specific proteins for destruction at the 26S-proteasome. These complexes are composed of three constant polypeptides--Skp1, Cullin1/3 and Roc1/Rbx1--and a fourth variable adapter, the F-box protein. Slimb (Slmb) is a Drosophila F-Box protein that fulfills several roles in development and cell physiology. We analyzed its participation in egg chamber development and found that slmb is required in both the follicle cells and the germline at different stages of oogenesis. We observed that in slmb somatic clones, morphogenesis of the germarium and encapsulation of the cyst were altered, giving rise to egg chambers with extra germline cells and two oocytes. Furthermore, in slmb somatic clones, we observed ectopic Fasciclin 3 expression, suggesting a delay in follicle cell differentiation, which correlated with the occurrence of ectopic polar cells, lack of interfollicular stalks and mislocalization of the oocyte. Later in oogenesis, Slmb was required in somatic cells to specify the position, size and morphology of dorsal appendages. Mild overactivation of the Dpp pathway caused similar phenotypes that could be antagonized by simultaneous overexpression of Slmb, suggesting that Slmb might normally downregulate the Dpp pathway in follicle cells. Indeed, ectopic expression of a dad-LacZ enhancer trap revealed that the Dpp pathway was upregulated in slmb somatic clones and, consistent with this, ectopic accumulation of the co-Smad protein, Medea, was recorded. By analyzing slmb germline clones, we found that loss of Slmb provoked a reduction in E2f2 and Dp levels, which correlated with misregulation of mitotic cycles during cyst formation, abnormal nurse cell endoreplication and impairment of dumping of the nurse cell content into the oocyte.     

Detection of somatic and germline mosaicism for the LAMP2 gene mutation c.808dupG in a Chinese family with Danon disease

Danon disease is a rare X-linked lysosomal storage disease characterized by hypertrophic cardiomyopathy, myopathy and mental retardation, and is due to a primary defect in lysosome-associated membrane protein-2 (LAMP 2). More than 26 mutations in the LAMP2 gene have been described, including a small number of de novo mutations, some of which are suspected to be caused by germline mosaicism. Here, we describe the first molecularly documented evidence of somatic mosaicism for a LAMP2 mutation, identified in the asymptomatic mother of a boy with Danon disease caused by the frameshift mutation c.808dupG (p.A270Gfx3) within exon 6. In addition, in order to gain insight into the possible explanation for the mother's lack of phenotype, the level of somatic mosaicism and the X-chromosome inactivation pattern were investigated. This study provides new insight into the causes of phenotypic variability in female mutation-carriers and underlines the importance of parental molecular testing for accurate genetic counseling for Danon disease.     

Slowmo is required for Drosophila germline proliferation

Null mutations in the Drosophila gene, slowmo (slmo), result in reduced mobility and lethality in first-instar larvae. Slowmo encodes a mitochondrial protein of unknown function, as do the two other homologs found in Drosophila. Here, we have studied a hypomorphic P-element allele of slmo demonstrating its effects on germline divisions in both testes and ovaries. Using in situ studies, enhancer-trap activity, and promoter fusions, we have shown that slmo expression in testes is found in the somatic cyst cells (SCC). The hypomorphic allele for Slmo revealed apoptotic loss of germline cells in the larval germline, culminating in a complete absence of the germline in adult flies. In females, a similar degeneration of the germarium is observed, while reporter gene expression is found in both germline and somatic cells. Using a null mutation in female germline clones, we find slmo is dispensable from the germline cells. Our results suggest that Slowmo is not required in germline cells directly, but is required in SCCs responsible for maintaining germline survival in both sexes.     

Interaction of N-ethyl-N'-nitro-n-nitrosoguanidine with nucleic acids and proteins in comparison with N-methyl-N'-nitro-N-nitrosoguanidine

Reactivity of N-ethyl-N'-nitro-N-nitrosoguanidine (ENNG) was studied in comparison with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). The radioactivity of [guanidino-14C]-ENNG was incorporated only into the protein fraction and that of [ethyl-14C]ENNG was incorporated into DNA, RNA and protein fractions in ascites hepatoma AH7974 cells, as were those of [guanidino-14C]- and [methyl-14C]MNNG, respectively. The amounts of the binding of ENNG were less than those of MNNG, especially in the corporation of the ethyl moiety of ENNG into nucleic acid fractions. In a non-cellular system, the radioactivity of [guanidino-14C]ENNG was incorporated into proteins, preferentially into basic proteins such as cytochrome c, but was not incorporated into nucleic acids. This behavior is similar to that of [guanidino-14C]MNNG, while the amount of binding of the former was about half of that of the latter. The radioactivity of [ethyl-14C]ENNG was also incorporated into basic proteins to almost the same extent as that of [methyl-14C]MNNG. However, the binding of the ethyl moiety of ENNG to nucleic acids was much lower than that of the methyl moiety of MNNG. Horse heart cytochrome c, bovine pancreatic RNase A and regenerating rat liver chromatin had altered their biological activities to various degrees after modification by ENNG or MNNG.     

Intact fetal ovarian cord formation promotes mouse oocyte survival and development

Background  

Female reproductive potential, or the ability to propagate life, is limited in mammals with the majority of oocytes lost before birth. In mice, surviving perinatal oocytes are enclosed in ovarian follicles for subsequent oocyte development and function in the adult. Before birth, fetal germ cells of both sexes develop in clusters, or germline cysts, in the undifferentiated gonad. Upon sex determination of the fetal gonad, germ cell cysts become organized into testicular or ovarian cord-like structures and begin to interact with gonadal somatic cells. Although germline cysts and testicular cords are required for spermatogenesis, the role of cyst and ovarian cord formation in mammalian oocyte development and female fertility has not been determined.     

Human Misato regulates mitochondrial distribution and morphology

Misato of Drosophila melanogaster and Saccharomyces cerevisiae DML1 are conserved proteins having a homologous region with a part of the GTPase family that includes eukaryotic tubulin and prokaryotic FtsZ. We characterized human Misato sharing homology with Misato of D. melanogaster and S. cerevisiae DML1. Tissue distribution of Misato exhibited ubiquitous distribution. Subcellular localization of the protein studied using anti-Misato antibody suggested that it is localized to the mitochondria. Further experiments of fractionating mitochondria revealed that Misato was localized to the outer membrane. The transfection of Misato siRNA led to growth deficiencies compared with control siRNA transfected HeLa cells, and the Misato-depleted HeLa cells showed apoptotic nuclear fragmentation resulting in cell death. After silencing of Misato, the filamentous mitochondrial network disappeared and fragmented mitochondria were observed, indicating human Misato has a role in mitochondrial fusion. To examine the effects of overexpression, COS-7 cells were transfected with cDNA encoding EGFP-Misato. Its overexpression resulted in the formation of perinuclear aggregations of mitochondria in these cells. The Misato-overexpressing cells showed low viability and had no nuclei or a small and structurally unusual ones. These results indicated that human Misato has a role(s) in mitochondrial distribution and morphology and that its unregulated expression leads to cell death.