Meredith Calvert

Early Depletion of Primordial Germ Cells in Zebrafish Promotes Testis Formation

Summary As complete absence of germ cells leads to sterile males in zebrafish, we explored the relationship between primordial germ cell (PGC) number and sexual development. Our results revealed dimorphic proliferation of PGCs in the early zebrafish larvae, marking the beginning of sexual differentiation. We applied morpholino-based gene knockdown and cell transplantation strategies to demonstrate that a threshold number of PGCs is required for the stability of ovarian fate. Using histology and transcriptomic analyses, we determined that zebrafish gonads are in a meiotic ovarian stage at 14 days postfertilization and identified signaling pathways supporting meiotic oocyte differentiation and eventual female fate. The development of PGC-depleted gonads appears to be restrained and delayed, suggesting that PGC number may directly regulate the variability and length of gonadal transformation and testicular differentiation in zebrafish. We propose that gonadal transformation may function as a developmental buffering mechanism to ensure the reproductive outcome.

Myosin concentration underlies cell size–dependent scalability of actomyosin ring constriction

The rate of actomyosin ring constriction in cells of different sizes correlates with myosin motor concentration in Neurospora crassa cells, leading to increased division rates in larger cells during cytokinesis.

The neuropeptide tachykinin is essential for pheromone detection in a gustatory neural circuit

Gustatory pheromones play an essential role in shaping the behavior of many organisms. However, little is known about the processing of taste pheromones in higher order brain centers. Here, we describe a male-specific gustatory circuit in Drosophila that underlies the detection of the anti-aphrodisiac pheromone (3R,11Z,19Z)-3-acetoxy-11,19-octacosadien-1-ol (CH503). Using behavioral analysis, genetic manipulation, and live calcium imaging, we show that Gr68a-expressing neurons on the forelegs of male flies exhibit a sexually dimorphic physiological response to the pheromone and relay information to the central brain via peptidergic neurons. The release of tachykinin from 8 to 10 cells within the subesophageal zone is required for the pheromone-triggered courtship suppression. Taken together, this work describes a neuropeptide-modulated central brain circuit that underlies the programmed behavioral response to a gustatory sex pheromone. These results will allow further examination of the molecular basis by which innate behaviors are modulated by gustatory cues and physiological state. DOI: http://dx.doi.org/10.7554/eLife.06914.001

The Golgi associated ERI3 is a Flavivirus host factor

Dengue virus (DENV) is a mosquito-borne Flavivirus classified into four serotypes (DENV-1-4) that causes Dengue fever (DF), Dengue hemorrhagic Fever (DHF) or Dengue shock syndrome (DSS). An estimated 390 million people are at risk for infection with DENV and there are no effective vaccines or therapeutics. We utilized RNA chromatography coupled with quantitative mass spectrometry (qMS) to identify host RNA binding proteins (RBPs) that interact with DENV-2 RNA. We identified ERI3 (also PRNPIP and PINT1), a putative 3′–5′ RNA exonuclease, which preferentially associates with DENV-2 genomic RNA via interactions with dumbbell structures in the 3′ UTR. ERI3 is required for accumulation of DENV-2 genomic RNA and production of infectious particles. Furthermore, the mosquito homologue of ERI3 is required for DENV-2 replication in adult Aedes aegypti mosquitos implying that the requirement for ERI3 is conserved in both DENV hosts. In human cells ERI3 localizes to the Golgi in uninfected cells, but relocalizes near sites of DENV-2 replication in infected cells. ERI3 is not required for maintaining DENV-2 RNA stability or translation of the viral polyprotein, but is required for viral RNA synthesis. Our results define a specific role for ERI3 and highlight the importance of Golgi proteins in DENV-2 replication.

Three-Dimensional Imaging of the Developing Human Fetal Urogenital-Genital Tract: Indifferent Stage to Male and Female Differentiation

Recent studies in our lab have utilized three imaging techniques to visualize the developing human fetal urogenital tract in three dimensions: optical projection tomography, scanning electron microscopy and lightsheet fluorescence microscopy. We have applied these technologies to examine changes in morphology and differential gene expression in developing human external genital specimens from the ambisexual stage (<9 weeks fetal age) to well-differentiated male and female organs (>13 weeks fetal age). This work outlines the history and function of each of these three imaging modalities, our methods to prepare specimens for each and the novel findings we have produced thus far. We believe the images in this paper of human fetal urogenital organs produced using lightsheet fluorescence microscopy are the first published to date.

New developments and novel applications in high throughput and high content imaging.

SINCE the first microscopes of Van Leeuwenhoek about 300 years ago, microscopy has continued to provide novel insights about the fundamental elements of life. Images are by nature rich in information and recent times have seen an increased effort to move beyond their descriptive value and to quantify this information. Over the past twenty-five years, significant advances in computational power, robotics and automation, and image detectors have made possible a huge increase in the throughput and content obtainable with light microscopy methods. Though the modalities that have been developed as platforms for high-content (HC) and high-throughput (HT) imaging vary somewhat, the resulting output is essentially the same: morphological data obtained from a large number of fields of cells. The term “high-throughput microscopy” first appeared in PubMed relatively recently (1) and in this case it referred to the use of an image-based screen of a cDNA library to identify genes that induced altered nuclear/cytoplasmic ratios of TORC1-eGFP. The following year, a chapter discussing the use of different screening strategies in RNAi based screens was the first use of the term “high-content microscopy” (2). In general, HC assays consider the morphological phenotypes of individual cells, rather than the average signals measured from all cells (3). Interestingly, the first example of imaging flow cytometry (IFC) appears much earlier, in 1979 (4). The authors developed a unit capable of detecting cellular particles, triggering a flashlamp if particles were in the preselected size range of interest, and feeding 16-mm film for acquisition, documentation, and image analysis. IFC has since been used to automate the analysis of many rare cellular events and those requiring data from large populations of cells, such as analysis of internalized bacteria (5), phagocytosis (6), nuclear–cytoplasmic translocation events (7), and yeast cell cycle analysis (8). Since this time the number of publications relying on HT/HC imaging methods has, predictably, grown exponentially (Fig. 1). This has led to a simultaneous explosion in the scale and content of image data and, most importantly, the type of scientific questions that can be asked when interrogating these datasets. These technological advances have brought imaging into the realm of “omics,” in which large datasets permit the quantification of molecular and morphological phenotypes that can inform as to the structure, function, and dynamics of a biological system. There are essentially four areas in which technological advances have had a significant impact on HT/HC imaging: (i) Reagents and assay tools, e.g. advanced fluorescent probes, genome-wide RNAi libraries, and gene editing techniques (e.g., TALENs and CRISPR/Cas9); (ii) Microscopy and optical advancements, e.g. high sensitivity detectors (sCMOS cameras and GaAsp detectors) and superresolution microscopy; (iii) Automation and robotics, such as the robotic microscope (9) and commercially available HT/HC platforms such as the Operetta (PerkinElmer) and the IN Cell Analyzer (GE Lifesciences); and (iv) Computational advancements, since one of the major bottlenecks for most HT/HC imaging platforms is how to handle storage, processing, and analysis of ever larger datasets. Improvements in the speed and capacity of desktop computing, tools for the rendering and analysis of three-dimensional image data, and platforms for archiving and analyzing large image databases (such as OMERO, Bisque, and Cell Profiler) have all facilitated this. In this special issue, we will provide a broad snapshot of

Organic cation transporter 1 (OCT1) modulates multiple cardiometabolic traits through effects on hepatic thiamine content

A constellation of metabolic disorders, including obesity, dysregulated lipids, and elevations in blood glucose levels, has been associated with cardiovascular disease and diabetes. Analysis of data from recently published genome-wide association studies (GWAS) demonstrated that reduced-function polymorphisms in the organic cation transporter, OCT1 (SLC22A1), are significantly associated with higher total cholesterol, low-density lipoprotein (LDL) cholesterol, and triglyceride (TG) levels and an increased risk for type 2 diabetes mellitus, yet the mechanism linking OCT1 to these metabolic traits remains puzzling. Here, we show that OCT1, widely characterized as a drug transporter, plays a key role in modulating hepatic glucose and lipid metabolism, potentially by mediating thiamine (vitamin B1) uptake and hence its levels in the liver. Deletion of Oct1 in mice resulted in reduced activity of thiamine-dependent enzymes, including pyruvate dehydrogenase (PDH), which disrupted the hepatic glucose–fatty acid cycle and shifted the source of energy production from glucose to fatty acids, leading to a reduction in glucose utilization, increased gluconeogenesis, and altered lipid metabolism. In turn, these effects resulted in increased total body adiposity and systemic levels of glucose and lipids. Importantly, wild-type mice on thiamine deficient diets (TDs) exhibited impaired glucose metabolism that phenocopied Oct1 deficient mice. Collectively, our study reveals a critical role of hepatic thiamine deficiency through OCT1 deficiency in promoting the metabolic inflexibility that leads to the pathogenesis of cardiometabolic disease.

Measuring Physiological Responses of Drosophila Sensory Neurons to Lipid Pheromones Using Live Calcium Imaging.

Unlike mammals, insects such as Drosophila have multiple taste organs. The chemosensory neurons on the legs, proboscis, wings and ovipositor of Drosophila express gustatory receptors(1,2), ion channels(3-6), and ionotropic receptors(7) that are involved in the detection of volatile and non-volatile sensory cues. These neurons directly contact tastants such as food, noxious substances and pheromones and therefore influence many complex behaviors such as feeding, egg-laying and mating. Electrode recordings and calcium imaging have been widely used in insects to quantify the neuronal responses evoked by these tastants. However, electrophysiology requires specialized equipment and obtaining measurements from a single taste sensillum can be technically challenging depending on the cell-type, size, and position. In addition, single neuron resolution in Drosophila can be difficult to achieve since taste sensilla house more than one type of chemosensory neuron. The live calcium imaging method described here allows responses of single gustatory neurons in live flies to be measured. This method is especially suitable for imaging neuronal responses to lipid pheromones and other ligand types that have low solubility in water-based solvents.