A High-Throughput Method for Quantifying Fecundity.

The fruit fly, , is an experimentally tractable model system that has recently emerged as a powerful "new approach methodology" (NAM) for chemical safety testing. As oogenesis is well conserved at the molecular and cellular level, measurements of fecundity can be useful for identifying chemicals that affect reproductive health across species. However, standard fecundity assays have been difficult to perform in a high-throughput manner because experimental factors such as the physiological state of the flies and environmental cues must be carefully controlled to achieve consistent results.

Arp2/3 complex activity enables nuclear YAP for naïve pluripotency of human embryonic stem cells.

Our understanding of the transitions of human embryonic stem cells (hESCs) between distinct stages of pluripotency relies predominantly on regulation by transcriptional and epigenetic programs with limited insight on the role of established morphological changes. We report remodeling of the actin cytoskeleton of hESCs as they transition from primed to naïve pluripotency which includes assembly of a ring of contractile actin filaments encapsulating colonies of naïve hESCs. Activity of the Arp2/3 complex is required for formation of the actin ring, to establish uniform cell mechanics within naïve colonies, to promote nuclear translocation of the Hippo pathway effectors YAP and TAZ, and for effective transition to naïve pluripotency.

PROPEL: a scalable model for postbaccalaureate training to promote diversity in the biomedical workforce.

Promoting diversity in the scientific workforce is crucial for harnessing the potential of available talent and ensuring equitable access to Science, Technology, Engineering, Mathematics, and Medicine (STEM-M) careers. We have developed an innovative program called Postbaccalaureate Research Opportunity to Promote Equity in Learning (PROPEL) that provides scientific and career development training for postbaccalaureate scholars from historically excluded backgrounds in STEM-M fields with an interest in pursuing a PhD or MD/PhD degree. Our program is distinct from other postbaccalaureate programs in that scholars are hired by individual labs rather than funded centrally by the program.

A high-throughput method for quantifying fecundity.

Measurements of Drosophila fecundity are used in a wide variety of studies, such as investigations of stem cell biology, nutrition, behavior, and toxicology. In addition, because fecundity assays are performed on live flies, they are suitable for longitudinal studies such as investigations of aging or prolonged chemical exposure. However, standard Drosophila fecundity assays have been difficult to perform in a high-throughput manner because experimental factors such as the physiological state of the flies and environmental cues must be carefully controlled to achieve consistent results.

Single-cell RNA sequencing identifies eggplant as a regulator of germ cell development in Drosophila.

Drosophila ovarian germline stem cells (GSCs) are a powerful model for stem cell research. In this study, we use single-cell RNA sequencing (scRNA-seq), an RNAi screen and bioinformatic analysis, to identify genes involved in germ cell differentiation, including 34 genes with upregulated expression during early germ cell development and 19 genes that may regulate germ cell differentiation. Among these, a gene we have named eggplant (eggpl) is highly expressed in GSCs and downregulated in early daughter cells.

Fly Cell Atlas: A single-nucleus transcriptomic atlas of the adult fruit fly.

For more than 100 years, the fruit fly has been one of the most studied model organisms. Here, we present a single-cell atlas of the adult fly, Tabula , that includes 580,000 nuclei from 15 individually dissected sexed tissues as well as the entire head and body, annotated to >250 distinct cell types. We provide an in-depth analysis of cell type-related gene signatures and transcription factor markers, as well as sexual dimorphism, across the whole animal.

A single-cell atlas and lineage analysis of the adult Drosophila ovary.

The Drosophila ovary is a widely used model for germ cell and somatic tissue biology. Here we use single-cell RNA-sequencing (scRNA-seq) to build a comprehensive cell atlas of the adult Drosophila ovary that contains transcriptional profiles for every major cell type in the ovary, including the germline stem cells and their niche cells, follicle stem cells, and previously undescribed subpopulations of escort cells. In addition, we identify Gal4 lines with specific expression patterns and perform lineage tracing of subpopulations of escort cells and follicle cells.

The follicle epithelium in the ovary is maintained by a small number of stem cells.

The follicle stem cells (FSCs) in the ovary are an important experimental model for the study of epithelial stem cell biology. Although decades of research support the conclusion that there are two FSCs per ovariole, a recent study used a novel clonal marking system to conclude that there are 15-16 FSCs per ovariole. We performed clonal analysis using both this novel clonal marking system and standard clonal marking systems, and identified several problems that may have contributed to the overestimate of FSC number.

Drosophila anion exchanger 2 is required for proper ovary development and oogenesis.

Understanding how cell fate decisions are regulated is a central question in stem cell biology. Recent studies have demonstrated that intracellular pH (pHi) dynamics contribute to this process. Indeed, the pHi of cells within a tissue is not simply a consequence of chemical reactions in the cytoplasm and other cellular activity, but is actively maintained at a specific setpoint in each cell type.

Wingless promotes EGFR signaling in follicle stem cells to maintain self-renewal.

Adult stem cell niche boundaries must be precisely maintained to facilitate the segregation of stem cell and daughter cell fates. However, the mechanisms that govern this process in epithelial tissues are not fully understood. In this study, we investigated the relationship between two signals, Wnt and EGFR, that are necessary for self-renewal of the epithelial follicle stem cells (FSCs) in the ovary, but must be downregulated in cells that have exited the niche to allow for differentiation.

Phosphorylated Groucho delays differentiation in the follicle stem cell lineage by providing a molecular memory of EGFR signaling in the niche.

In the epithelial follicle stem cells (FSCs) of the Drosophila ovary, Epidermal Growth Factor Receptor (EGFR) signaling promotes self-renewal, whereas Notch signaling promotes differentiation of the prefollicle cell (pFC) daughters. We have identified two proteins, Six4 and Groucho (Gro), that link the activity of these two pathways to regulate the earliest cell fate decision in the FSC lineage. Our data indicate that Six4 and Gro promote differentiation towards the polar cell fate by promoting Notch pathway activity.

Increased intracellular pH is necessary for adult epithelial and embryonic stem cell differentiation.

Despite extensive knowledge about the transcriptional regulation of stem cell differentiation, less is known about the role of dynamic cytosolic cues. We report that an increase in intracellular pH (pHi) is necessary for the efficient differentiation of Drosophila adult follicle stem cells (FSCs) and mouse embryonic stem cells (mESCs). We show that pHi increases with differentiation from FSCs to prefollicle cells (pFCs) and follicle cells.

EGFR signaling promotes self-renewal through the establishment of cell polarity in Drosophila follicle stem cells.

Epithelial stem cells divide asymmetrically, such that one daughter replenishes the stem cell pool and the other differentiates. We found that, in the epithelial follicle stem cell (FSC) lineage of the Drosophila ovary, epidermal growth factor receptor (EGFR) signaling functions specifically in the FSCs to promote the unique partially polarized state of the FSC, establish apical-basal polarity throughout the lineage, and promote FSC maintenance in the niche. In addition, we identified a novel connection between EGFR signaling and the cell-polarity regulator liver kinase B1 (LKB1), which indicates that EGFR signals through both the Ras-Raf-MEK-Erk pathway and through the LKB1-AMPK pathway to suppress apical identity.

Basolateral junction proteins regulate competition for the follicle stem cell niche in the Drosophila ovary.

Epithelial stem cells are routinely lost or damaged during adult life and must therefore be replaced to maintain homeostasis. Recent studies indicate that stem cell replacement occurs through neutral competition in many types of epithelial tissues, but little is known about the factors that determine competitive outcome. The epithelial follicle stem cells (FSCs) in the Drosophila ovary are regularly lost and replaced during normal homeostasis, and we show that FSC replacement conforms to a model of neutral competition.

A dynamic population of stromal cells contributes to the follicle stem cell niche in the Drosophila ovary.

Epithelial stem cells are maintained within niches that promote self-renewal by providing signals that specify the stem cell fate. In the Drosophila ovary, epithelial follicle stem cells (FSCs) reside in niches at the anterior tip of the tissue and support continuous growth of the ovarian follicle epithelium. Here, we demonstrate that a neighboring dynamic population of stromal cells, called escort cells, are FSC niche cells.

Drosophila models of epithelial stem cells and their niches.

Epithelial stem cells are regulated through a complex interplay of signals from diffusible ligands, cellular interactions, and attachment to the extracellular matrix. The development of Drosophila models of epithelial stem cells and their associated niche has made it possible to dissect the contribution of each of these factors in vivo, during both basal homeostasis and in response to acute damage such as infection. Studies of Drosophila epithelial stem cells have also provided insight into the mechanisms by which a healthy population of stem cells are maintained throughout adulthood by demonstrating, for example, that stem cells have a finite lifespan and may be displaced by replacement cells competing for niche occupancy.

The carnegie protein trap library: a versatile tool for Drosophila developmental studies.

Metazoan physiology depends on intricate patterns of gene expression that remain poorly known. Using transposon mutagenesis in Drosophila, we constructed a library of 7404 protein trap and enhancer trap lines, the Carnegie collection, to facilitate gene expression mapping at single-cell resolution. By sequencing the genomic insertion sites, determining splicing patterns downstream of the enhanced green fluorescent protein (EGFP) exon, and analyzing expression patterns in the ovary and salivary gland, we found that 600-900 different genes are trapped in our collection.

Breaking out of the mold: diversity within adult stem cells and their niches.

During the past several years, it has become increasingly possible to study adult stem cells in their native territories within tissues. These studies have provided new evidence for the existence of stem cells in the breast, muscle, lung and kidney and have led to a deeper understanding of the best-known stem cells in Drosophila and mice. Tissue stem cells are turning out to be diverse, with varying division rates, lineage lengths, and mechanisms of regulation.

Carbon monoxide-induced suspended animation protects against hypoxic damage in Caenorhabditis elegans.

Oxygen deprivation is a major cause of cellular damage and death. Here we demonstrate that Caenorhabditis elegans embryos, which can survive both in anoxia (<0.001 kPa O(2)) by entering into suspended animation and in mild hypoxia (0.

Suspended animation in C. elegans requires the spindle checkpoint.

In response to environmental signals such as anoxia, many organisms enter a state of suspended animation, an extreme form of quiescence in which microscopically visible movement ceases. We have identified a gene, san-1, that is required for suspended animation in Caenorhabditis elegans embryos. We show that san-1 functions as a spindle checkpoint component in C.

Dephosphorylation of cell cycle-regulated proteins correlates with anoxia-induced suspended animation in Caenorhabditis elegans.

Some metazoans have evolved the capacity to survive severe oxygen deprivation. The nematode, Caenorhabditis elegans, exposed to anoxia (0 kPa, 0% O(2)) enters into a recoverable state of suspended animation during all stages of the life cycle. That is, all microscopically observable movement ceases including cell division, developmental progression, feeding, and motility.