Studies of the Drosophila ovary have provided significant insight into the molecular and cellular processes that control cell function, tissue organization, and animal development. To characterize ...mutants with defects in oogenesis or to observe the distribution of gene products involved in egg production, the ovaries must be carefully extracted and prepared for analysis. This chapter describes the manual dissection of ovaries from adult Drosophila females, followed by standard fixation and staining of the isolated tissue. Specifically, this chapter provides procedures for simple DNA and F-actin staining to assess cell and tissue morphology, as well as immunostaining to localize proteins of interest.
Gametes are highly specialized cell types produced by a complex differentiation process. Production of viable oocytes requires a series of precise and coordinated molecular events. Early in their ...development, germ cells are an interconnected group of mitotically dividing cells. Key regulatory events lead to the specification of mature oocytes and initiate a switch to the meiotic cell cycle program. Though the chromosomal events of meiosis have been extensively studied, it is unclear how other aspects of oocyte specification are temporally coordinated. The fruit fly,
, has long been at the forefront as a model system for genetics and cell biology research. The adult
ovary continuously produces germ cells throughout the organism's lifetime, and many of the cellular processes that occur to establish oocyte fate are conserved with mammalian gamete development. Here, we review recent discoveries from
that advance our understanding of how early germ cells balance mitotic exit with meiotic initiation. We discuss cell cycle control and establishment of cell polarity as major themes in oocyte specification. We also highlight a germline-specific organelle, the fusome, as integral to the coordination of cell division, cell polarity, and cell fate in ovarian germ cells. Finally, we discuss how the molecular controls of the cell cycle might be integrated with cell polarity and cell fate to maintain oocyte production.
The Fly-CURE is a genetics-focused multi-institutional Course-Based Undergraduate Research Experience (CURE) that provides undergraduate students with hands-on research experiences within a course. ...Through the Fly-CURE, undergraduate students at diverse types of higher education institutions across the United States map and characterize novel mutants isolated from a genetic screen in
. To date, more than 20 mutants have been studied across 20 institutions, and our scientific data have led to eleven publications with more than 500 students as authors. To evaluate the impact of the Fly-CURE experience on students, we developed and validated assessment tools to identify students' perceived research self-efficacy, sense of belonging in science, and intent to pursue additional research opportunities. Our data, collected over three academic years and involving 14 institutions and 480 students, show gains in these metrics after completion of the Fly-CURE across all student subgroups analyzed, including comparisons of gender, academic status, racial and ethnic groups, and parents' educational background. Importantly, our data also show differential gains in the areas of self-efficacy and interest in seeking additional research opportunities between Fly-CURE students with and without prior research experience, illustrating the positive impact of research exposure (dosage) on student outcomes. Altogether, our data indicate that the Fly-CURE experience has a significant impact on students' efficacy with research methods, sense of belonging to the scientific research community, and interest in pursuing additional research experiences.
We previously showed that asunder (asun) is a critical regulator of dynein localization during Drosophila spermatogenesis. Because the expression of asun is much higher in Drosophila ovaries and ...early embryos than in testes, we herein sought to determine whether ASUN plays roles in oogenesis and/or embryogenesis. We characterized the female germline phenotypes of flies homozygous for a null allele of asun (asund93). We find that asund93 females lay very few eggs and contain smaller ovaries with a highly disorganized arrangement of ovarioles in comparison to wild-type females. asund93 ovaries also contain a significant number of egg chambers with structural defects. A majority of the eggs laid by asund93 females are ventralized to varying degrees, from mild to severe; this ventralization phenotype may be secondary to defective localization of gurken transcripts, a dynein-regulated step, within asund93 oocytes. We find that dynein localization is aberrant in asund93 oocytes, indicating that ASUN is required for this process in both male and female germ cells. In addition to the loss of gurken mRNA localization, asund93 ovaries exhibit defects in other dynein-mediated processes such as migration of nurse cell centrosomes into the oocyte during the early mitotic divisions, maintenance of the oocyte nucleus in the anterior-dorsal region of the oocyte in late-stage egg chambers, and coupling between the oocyte nucleus and centrosomes. Taken together, our data indicate that asun is a critical regulator of dynein localization and dynein-mediated processes during Drosophila oogenesis.
•Females null for asun (asund93) have a severe egg-laying defect.•asund93-derived embryos are ventralized due to mislocalization of grk transcripts.•Dynein, required for grk localization, is mislocalizaed in asund93 egg chambers.•asund93 ovaries exhibit defects in known dynein-mediated processes.•ASUN regulates dynein and dynein-mediated processes during Drosophila oogenesis.
We previously identified a Drosophila maternal effect-lethal mutant named 'no poles' (nopo). Embryos from nopo females undergo mitotic arrest with barrel-shaped, acentrosomal spindles during the ...rapid cycles of syncytial embryogenesis because of activation of a Chk2-mediated DNA checkpoint. NOPO is the Drosophila homolog of human TNF receptor associated factor (TRAF)-interacting protein (TRIP), which has been implicated in TNF signaling. NOPO and TRIP contain RING domains closely resembling those of known E3 ubiquitin ligases. We herein sought to elucidate the mechanism by which TRIP/NOPO promotes genomic stability by performing a yeast two-hybrid screen to identify potential substrates/interactors. We identified members of the Y-family of DNA polymerases that facilitate replicative bypass of damaged DNA (translesion synthesis) as TRIP interactors. We show that TRIP and NOPO co-immunoprecipitate with human and Drosophila Polη, respectively, from cultured cells. We generated a null mutation in Drosophila Polη (dPolη) and found that dPolη-derived embryos have increased sensitivity to ultraviolet irradiation and exhibit nopo-like mitotic spindle defects. dPolη and nopo interact genetically in that overexpression of dPolη in hypomorphic nopo-derived embryos suppresses nopo phenotypes. We observed enhanced ubiquitylation of Polη by TRIP and NOPO E3 ligases in human cells and Drosophila embryos, respectively, and show that TRIP promotes hPolη localization to nuclear foci in human cells. We present a model in which TRIP/NOPO ubiquitylates Polη to positively regulate its activity in translesion synthesis.
We present the complete genome sequences of two viruses with siphovirus morphology, isolated from soils collected in Southwestern Indiana using the host
. Spelly is a BE2 cluster phage with a ...131,347-bp genome. Phredrick is a BK1 cluster phage with a 128,873-bp genome.
In a screen for cell-cycle regulators, we identified a Drosophila maternal effect-lethal mutant that we named ` no poles ' ( nopo ). Embryos from nopo females undergo mitotic arrest with ...barrel-shaped, acentrosomal spindles during the rapid S-M cycles of syncytial embryogenesis. We identified CG5140 , which encodes a candidate RING domain-containing E3 ubiquitin ligase, as the nopo gene. A conserved residue in the RING domain is altered in our EMS-mutagenized allele of nopo , suggesting that E3 ligase activity is crucial for NOPO function. We show that mutation of a DNA checkpoint kinase, CHK2, suppresses the spindle and developmental defects of nopo -derived embryos, revealing that activation of a DNA checkpoint operational in early embryos contributes significantly to the nopo phenotype. CHK2-mediated mitotic arrest has been previously shown to occur in response to mitotic entry with DNA damage or incompletely replicated DNA. Syncytial embryos lacking NOPO exhibit a shorter interphase during cycle 11, suggesting that they may enter mitosis prior to the completion of DNA replication. We show that Bendless (BEN), an E2 ubiquitin-conjugating enzyme, interacts with NOPO in a yeast two-hybrid assay; furthermore, ben -derived embryos arrest with a nopo -like phenotype during syncytial divisions. These data support our model that an E2-E3 ubiquitination complex consisting of BEN-UEV1A (E2 heterodimer) and NOPO (E3 ligase) is required for the preservation of genomic integrity during early embryogenesis.
Cdc14 is an evolutionarily conserved serine/threonine phosphatase. Originally identified in
as a cell cycle regulator, its role in other eukaryotic organisms remains unclear. In
, Cdc14 is encoded by ...a single gene, thus facilitating its study. We found that Cdc14 expression is highest in the testis of adult flies and that
null flies are viable.
null female and male flies do not display altered fertility.
null males, however, exhibit decreased sperm competitiveness. Previous studies have shown that Cdc14 plays a role in ciliogenesis during zebrafish development. In Drosophila, sensory neurons are ciliated. We found that the Drosophila
null mutants have defects in chemosensation and mechanosensation as indicated by decreased avoidance of repellant substances and decreased response to touch. In addition, we show that
null mutants have defects in lipid metabolism and resistance to starvation. These studies highlight the diversity of Cdc14 function in eukaryotes despite its structural conservation.
Meat and poultry processing facilities face distinctive challenges in the control of infectious diseases, including coronavirus disease 2019 (COVID-19) (1). COVID-19 outbreaks among meat and poultry ...processing facility workers can rapidly affect large numbers of persons. Assessment of COVID-19 cases among workers in 115 meat and poultry processing facilities through April 27, 2020, documented 4,913 cases and 20 deaths reported by 19 states (1). This report provides updated aggregate data from states regarding the number of meat and poultry processing facilities affected by COVID-19, the number and demographic characteristics of affected workers, and the number of COVID-19-associated deaths among workers, as well as descriptions of interventions and prevention efforts at these facilities. Aggregate data on confirmed COVID-19 cases and deaths among workers identified and reported through May 31, 2020, were obtained from 239 affected facilities (those with a laboratory-confirmed COVID-19 case in one or more workers) in 23 states.* COVID-19 was confirmed in 16,233 workers, including 86 COVID-19-related deaths. Among 14 states reporting the total number of workers in affected meat and poultry processing facilities (112,616), COVID-19 was diagnosed in 9.1% of workers. Among 9,919 (61%) cases in 21 states with reported race/ethnicity, 87% occurred among racial and ethnic minority workers. Commonly reported interventions and prevention efforts at facilities included implementing worker temperature or symptom screening and COVID-19 education, mandating face coverings, adding hand hygiene stations, and adding physical barriers between workers. Targeted workplace interventions and prevention efforts that are appropriately tailored to the groups most affected by COVID-19 are critical to reducing both COVID-19-associated occupational risk and health disparities among vulnerable populations. Implementation of these interventions and prevention efforts
across meat and poultry processing facilities nationally could help protect workers in this critical infrastructure industry.
Mutant
B.4.1
, generated via EMS mutagenesis in
Drosophila
melanogaster
, was studied by undergraduate students participating in the Fly-CURE. After inducing genetically mosaic tissue in the adult ...eye,
B.4.1
mutant tissue displays a robust increase in cell division and a rough appearance. Complementation mapping and sequence analysis identified a nonsense mutation in the gene
CG1603
, which we named
clifford
(
cliff
) due to observed increases in red-pigmented mutant tissue compared to controls.
cliff
encodes a zinc finger-containing protein implicated in transcriptional control. RNAi knockdown of
cliff
similarly results in rough eyes, confirming a role for Cliff in eye development.
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