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.

Intracellular pH dynamics regulates intestinal stem cell lineage specification.

Intracellular pH dynamics is increasingly recognized to regulate myriad cell behaviors. We report a finding that intracellular pH dynamics also regulates adult stem cell lineage specification. We identify an intracellular pH gradient in mouse small intestinal crypts, lowest in crypt stem cells and increasing along the crypt column.

Preparation of Drosophila Ovarioles for Single-Cell RNA Sequencing.

The production of eggs in the Drosophila ovary requires complex interactions between multiple cell types that coexist within the same solid tissue. This cellular heterogeneity makes the ovary a rich subject of study, but also makes it challenging to identify transcriptional differences between individual cell types using methods such as bulk RNA sequencing. The development of single-cell RNA sequencing (scRNA-seq) techniques addresses this limitation by providing an avenue to profile genetic and functional heterogeneity at a cellular resolution.

A DLG1-ARHGAP31-CDC42 axis is essential for the intestinal stem cell response to fluctuating niche Wnt signaling.

A central factor in the maintenance of tissue integrity is the response of stem cells to variations in the levels of niche signals. In the gut, intestinal stem cells (ISCs) depend on Wnt ligands for self-renewal and proliferation. Transient increases in Wnt signaling promote regeneration after injury or in inflammatory bowel diseases, whereas constitutive activation of this pathway leads to colorectal cancer.

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.

Distinct roles of Bendless in regulating FSC niche competition and daughter cell differentiation.

A major goal in the study of adult stem cells is to understand how cell fates are specified at the proper time and place to facilitate tissue homeostasis. Here, we found that an E2 ubiquitin ligase, Bendless (Ben), has multiple roles in the Drosophila ovarian epithelial follicle stem cell (FSC) lineage. First, Ben is part of the JNK signaling pathway, and we found that it, as well as other JNK pathway genes, are essential for differentiation of FSC daughter cells.

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.

Signal transduction in the early Drosophila follicle stem cell lineage.

The follicle stem cell (FSC) lineage in the Drosophila ovary is a highly informative model of in vivo epithelial stem cell biology. Studies over the past 30 years have identified roles for every major signaling pathway in the early FSC lineage. These pathways regulate a wide variety of cell behaviors, including self-renewal, proliferation, survival and differentiation.

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.

Cell fate decisions: emerging roles for metabolic signals and cell morphology.

Understanding how cell fate decisions are regulated is a fundamental goal of developmental and stem cell biology. Most studies on the control of cell fate decisions address the contributions of changes in transcriptional programming, epigenetic modifications, and biochemical differentiation cues. However, recent studies have found that other aspects of cell biology also make important contributions to regulating cell fate decisions.

Methods for Imaging Intracellular pH of the Follicle Stem Cell Lineage in Live Drosophila Ovarian Tissue.

Changes in intracellular pH (pHi) play important roles in the regulation of many cellular functions, including metabolism, proliferation, and differentiation. Typically, pHi dynamics are determined in cultured cells, which are amenable to measuring and experimentally manipulating pHi. However, the recent development of new tools and methodologies has made it possible to study pHi dynamics within intact, live tissue.

Neutral Competition for Follicle and Cyst Stem Cell Niches Requires Vesicle Trafficking Genes.

The process of selecting for cellular fitness through competition plays a critical role in both development and disease. The germarium, a structure at the tip of the ovariole of a ovary, contains two follicle stem cells (FSCs) that undergo neutral competition for the stem cell niche. Using the FSCs as a model, we performed a genetic screen through a collection of 126 mutants in essential genes on the chromosome to identify candidates that increase or decrease competition for the FSC niche.

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.

A Pak-regulated cell intercalation event leading to a novel radial cell polarity is involved in positioning of the follicle stem cell niche in the Drosophila ovary.

In the germarium of the Drosophila ovary, germline cysts are encapsulated one at a time by a follicular epithelium derived from two follicle stem cells (FSCs). Ovaries in flies mutant for the serine/threonine kinase Pak exhibit a novel phenotype, in which two side-by-side cysts are encapsulated at a time, generating paired egg chambers. This striking phenotype originates in the pupal ovary, where the developing germarium is shaped by the basal stalk, a stack of cells formed by cell intercalation.

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.

Enhancer-trap flippase lines for clonal analysis in the Drosophila ovary.

The Drosophila melanogaster genetic tool box includes many stocks for generating genetically mosaic tissue in which a clone of cells, related by lineage, contain a common genetic alteration. These tools have made it possible to study the postembryonic function of essential genes and to better understand how individual cells interact within intact tissues. We have screened through 201 enhancer-trap flippase lines to identify lines that produce useful clone patterns in the adult ovary.

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.