Whether or not mammalian females generate new oocytes during adulthood from germ-line stem cells to sustain the ovarian follicle pool has recently generated controversy. We used a sensitive lineage-labeling system to determine whether stem cells are needed in female adult mice to compensate for follicular losses and to directly identify active germ-line stem cells. Primordial follicles generated during fetal life are highly stable, with a half-life during adulthood of 10 mo, and thus are sufficient to sustain adult oogenesis without a source of renewal. Moreover, in normal mice or following germ-cell depletion with Busulfan, only stable, single oocytes are lineage-labeled, rather than cell clusters indicative of new oocyte formation. Even one germline stem cell division per 2 wk would have been detected by our method, based on the kinetics of fetal follicle formation. Thus, adult female mice neither require nor contain active germ-line stem cells or produce new oocytes in vivo.

Mammalian germ cells divide mitotically and form nests of associated cells just prior to entering meiosis. At least some nests contain germline cysts that arise by synchronous, incomplete mitotic divisions, but others may form by aggregation. To systematically investigate early murine germ cell development, we lineage marked the progeny of individual, newly arrived primordial germ cells in the E10.5 gonad. All the marked germ cells initially develop into clones containing two, four or eight cells, indicating cyst formation. Surprisingly, growing cysts in both sexes partially fragment into smaller cysts prior to completion and associate with cysts from unrelated progenitors. At the time divisions cease, female clones comprise five cysts on average that eventually give rise to about six primordial follicles. Male cyst cells break apart and probably become spermatogonial stem cells. Thus, cysts are invariant units of mouse germ cell development and cyst fragmentation provides insight into the amplification of spermatogonial stem cells and the origin of primordial follicles.

How oocytes are transferred into an oviduct with a receptive environment remains poorly known. We found that glands of the Drosophila female reproductive tract, spermathecae and/or parovaria, are required for ovulation and to promote sperm storage. Reducing total secretory cell number by interferring with Notch signaling during development blocked ovulation. Knocking down expression after adult eclosion of the nuclear hormone receptor Hr39, a master regulator of gland development, slowed ovulation and blocked sperm storage. However, ovulation (but not sperm storage) continued when only canonical protein secretion was compromised in adult glands. Our results imply that proteins secreted during adulthood by the canonical secretory pathway from female reproductive glands are needed to store sperm, while a non-canonical glandular secretion stimulates ovulation. Our results suggest that the reproductive tract signals to the ovary using glandular secretions, and that this pathway has been conserved during evolution.

Background: Reestablishing epithelial integrity and biosynthetic capacity is critically important following tissue damage. The adult Drosophila abdominal epithelium provides an attractive new system to address how postmitotic diploid cells contribute to repair.

DNA replication remains unfinished in many Drosophila polyploid cells, which harbor disproportionately fewer copies of late-replicating chromosomal regions. By analyzing paired-end high-throughput sequence data from polytene larval salivary gland cells, we define 112 underreplicated (UR) euchromatic regions 60-480 kb in size. To determine the effects of underreplication on genome integrity, we analyzed anomalous read pairs and breakpoint reads throughout the euchromatic genome. Each UR euchromatic region contains many different deletions 10-500 kb in size, while very few deletions are present in fully replicated chromosome regions or UR zones from embryo DNA. Thus, during endocycles, stalled forks within UR regions break and undergo local repair instead of remaining stable and generating nested forks. As a result, each salivary gland cell contains hundreds of unique deletions that account for their copy number reductions. Similar UR regions and deletions were observed in ovarian DNA, suggesting that incomplete replication, fork breakage, and repair occur widely in polytene cells. UR regions are enriched in genes encoding immunoglobulin superfamily proteins and contain many neurally expressed and homeotic genes. We suggest that the extensive somatic DNA instability described here underlies position effect variegation, molds the structure of polytene chromosomes, and should be investigated for possible functions.

Progenitors are early lineage cells that proliferate before the onset of terminal differentiation. Although widespread, the epigenetic mechanisms that control the progenitor state and the onset of differentiation remain elusive. By studying Drosophila ovarian follicle cell progenitors, we identified lysine-specific demethylase 1 (lsd1) and CoRest as differentiation regulators using a GAL4::GFP variegation assay. The follicle cell progenitors in lsd1 or CoRest heterozygotes prematurely lose epigenetic plasticity, undergo the Notch-dependent mitotic-endocycle transition, and stop dividing before a normal number of follicle cells can be produced. Simultaneously reducing the dosage of the histone H3K4 methyltransferase Trithorax reverses these effects, suggesting that an Lsd1/CoRest complex times progenitor differentiation by controlling the stability of H3K4 methylation levels. Individual cells or small clones initially respond to Notch; hence, a critical level of epigenetic stabilization is acquired cell-autonomously and initiates differentiation by making progenitors responsive to pre-existing external signals.

We found that the midgut shows striking regional differentiation along its anterior-posterior axis. Ten distinct subregions differ in cell morphology, gene expression and aspects of Notch signaling. RNA from isolated regions that was analyzed by RNAseq revealed spatially regulated expression of hundreds of enzymes and other genes with likely tissue functions.

Here, we document a collection of similar to 7434 MiMIC (Minos Mediated Integration Cassette) insertions of which 2854 are inserted in coding introns. They allowed us to create a library of 400 GFP-tagged genes. We show that 72% of internally tagged proteins are functional, and that more than 90% can be imaged in unfixed tissues. Moreover, the tagged mRNAs can be knocked down by RNAi against GFP (iGFPi), and the tagged proteins can be efficiently knocked down by deGradFP technology. The phenotypes associated with RNA and protein knockdown typically correspond to severe loss of function or null mutant phenotypes. Finally, we demonstrate reversible, spatial, and temporal knockdown of tagged proteins in larvae and adult flies. This new strategy and collection of strains allows unprecedented in vivo manipulations in flies for many genes. These strategies will likely extend to vertebrates.

Reproduction is heavily influenced by nutrition and metabolic state. Many common reproductive disorders in humans are associated with diabetes and metabolic syndrome. We characterized the metabolic mechanisms that support oogenesis and found that mitochondria in mature Drosophila oocytes enter a low-activity state of respiratory quiescence by remodeling the electron transport chain (ETC). This shift in mitochondrial function leads to extensive glycogen accumulation late in oogenesis and is required for the developmental competence of the oocyte. Decreased insulin signaling initiates ETC remodeling and mitochondrial respiratory quiescence through glycogen synthase kinase 3 (GSK3). Intriguingly, we observed similar ETC remodeling and glycogen uptake in maturing Xenopus oocytes, suggesting that these processes are evolutionarily conserved aspects of oocyte development. Our studies reveal an important link between metabolism and oocyte maturation.

Tissue integrity and homeostasis often rely on the proliferation of stem cells or differentiated cells to replace lost, aged, or damaged cells. Recently, we described an alternative source of cell replacement-the expansion of resident, non-dividing diploid cells by wound-induced polyploidization (WIP). Here we show that the magnitude of WIP is proportional to the extent of cell loss using a new semi-automated assay with single cell resolution. Hippo and JNK signaling regulate WIP; unexpectedly however, JNK signaling through AP-1 limits rather than stimulates the level of Yki activation and polyploidization in the Drosophila epidermis. We found that polyploidization also quantitatively compensates for cell loss in a mammalian tissue, mouse corneal endothelium, where increased cell death occurs with age in a mouse model of Fuchs Endothelial Corneal Dystrophy (FECD). Our results suggest that WIP is an evolutionarily conserved homeostatic mechanism that maintains the size and synthetic capacity of adult tissues.