Auxin exposure disrupts feeding behavior and fatty acid metabolism in adult Drosophila.

The ease of genetic manipulation in Drosophila melanogaster using the Gal4/UAS system has been beneficial in addressing key biological questions. Current modifications of this methodology to temporally induce transgene expression require temperature changes or exposure to exogenous compounds, both of which have been shown to have detrimental effects on physiological processes. The recently described auxin-inducible gene expression system (AGES) utilizes the plant hormone auxin to induce transgene expression and is proposed to be the least toxic compound for genetic manipulation, with no obvious effects on Drosophila development and survival in one wild-type strain. Here, we show that auxin delays larval development in another widely used fly strain, and that short- and long-term auxin exposure in adult Drosophila induces observable changes in physiology and feeding behavior. We further reveal a dosage response to adult survival upon auxin exposure, and that the recommended auxin concentration for AGES alters feeding activity. Furthermore, auxin-fed male and female flies exhibit a significant decrease in triglyceride levels and display altered transcription of fatty acid metabolism genes. Although fatty acid metabolism is disrupted, auxin does not significantly impact adult female fecundity or progeny survival, suggesting AGES may be an ideal methodology for studying limited biological processes. These results emphasize that experiments using temporal binary systems must be carefully designed and controlled to avoid confounding effects and misinterpretation of results.

Auxin Exposure Disrupts Feeding Behavior and Fatty Acid Metabolism in Adult Drosophila.

The ease of genetic manipulation in Drosophila melanogaster using the Gal4/UAS system has been beneficial in addressing key biological questions. Current modifications of this methodology to temporally induce transgene expression require temperature changes or exposure to exogenous compounds, both of which have been shown to have detrimental effects on physiological processes. The recently described auxin-inducible gene expression system (AGES) utilizes the plant hormone auxin to induce transgene expression and is proposed to be the least toxic compound for genetic manipulation, with no obvious effects on Drosophila development and survival in one wild-type strain. Here we show that auxin delays larval development in another widely-used fly strain, and that short- and long-term auxin exposure in adult Drosophila induces observable changes in physiology and feeding behavior. We further reveal a dosage response to adult survival upon auxin exposure, and that the recommended auxin concentration for AGES alters feeding activity. Furthermore, auxin fed male and female flies exhibit a significant decrease in triglyceride levels and display altered transcription of fatty acid metabolism genes. Although fatty acid metabolism is disrupted, auxin does not significantly impact adult female fecundity or progeny survival, suggesting AGES may be an ideal methodology for studying limited biological processes. These results emphasize that experiments using temporal binary systems must be carefully designed and controlled to avoid confounding effects and misinterpretation of results.

Analysis of Physiological Control of Adult Drosophila Oogenesis by Interorgan Communication.

Tissue homeostasis is dependent on the interaction between various organs within an organism in response to physiological inputs. The adult Drosophila melanogaster ovary is sensitive to environmental challenges and has recently been shown to be regulated by signaling from peripheral organs. To dissect the intricate coordination between overall organism health and reproduction, it is necessary to meticulously characterize both experimental tools and oogenesis processes. This chapter provides a guide for the careful analysis of interorgan communication in regulating oogenesis in adult Drosophila melanogaster.

Hormone receptor 4 is required in muscles and distinct ovarian cell types to regulate specific steps of Drosophila oogenesis.

The conserved nuclear receptor superfamily has crucial roles in many processes, including reproduction. Nuclear receptors with known roles in oogenesis have been studied mostly in the context of their ovary-intrinsic requirement. Recent studies in Drosophila, however, have begun to reveal new roles of nuclear receptor signaling in peripheral tissues in controlling reproduction. Here, we identified Hormone receptor 4 (Hr4) as an oogenesis regulator required in the ovary and muscles. Global Hr4 knockdown leads to increased germline stem cell (GSC) loss, reduced GSC proliferation, early germline cyst death, slowed follicle growth and vitellogenic follicle degeneration. Tissue-specific knockdown experiments uncovered ovary-intrinsic and peripheral tissue requirements for Hr4 In the ovary, Hr4 is required in the niche for GSC proliferation and in the germline for GSC maintenance. Hr4 functions in muscles to promote GSC maintenance and follicle growth. The specific tissues that require Hr4 for survival of early germline cysts and vitellogenic follicles remain unidentified. These results add to the few examples of muscles controlling gametogenesis and expand our understanding of the complexity of nuclear receptor regulation of various aspects of oogenesis.

Introductions to the Community: Early-Career Researchers in the Time of COVID-19.

COVID-19 has unfortunately halted lab work, conferences, and in-person networking, which is especially detrimental to researchers just starting their labs. Through social media and our reviewer networks, we met some early-career stem cell investigators impacted by the closures. Here, they introduce themselves and their research to our readers.

The Nuclear Receptor Seven Up Regulates Genes Involved in Immunity and Xenobiotic Response in the Adult Drosophila Female Fat Body.

The physiology of organisms depends on inter-organ communication in response to changes in the environment. Nuclear receptors are broadly expressed transcription factors that respond to circulating molecules to control many biological processes, including immunity, detoxification, and reproduction. Although the tissue-intrinsic roles of nuclear receptors in reproduction have been extensively studied, there is increasing evidence that nuclear receptor signaling in peripheral tissues can also influence oogenesis. We previously showed that the Drosophila nuclear receptor Seven up (Svp) is required in the adult fat body to regulate distinct steps of oogenesis; however, the relevant downstream targets of Svp remain unknown. Here, we took an RNA sequencing approach to identify candidate Svp targets specifically in the adult female fat body that might mediate this response. svp knockdown in the adult female fat body significantly downregulated immune genes involved in the first line of pathogen defense, suggesting a role for Svp in stimulating early immunity. In addition, we found that Svp transcriptionally regulates genes involved in each step of the xenobiotic detoxification response. Based on these findings, we propose a testable model in which Svp functions in the adult female fat body to stimulate early defense against pathogens and facilitate detoxification as part of its mechanisms to promote oogenesis.

Analysis of Gal4 Expression Patterns in Adult Drosophila Females.

Precise genetic manipulation of specific cell types or tissues to pinpoint gene function requirement is a critical step in studies aimed at unraveling the intricacies of organismal physiology. Drosophila researchers heavily rely on the UAS/Gal4/Gal80 system for tissue-specific manipulations; however, it is often unclear whether the reported Gal4 expression patterns are indeed specific to the tissue of interest such that experimental results are not confounded by secondary sites of Gal4 expression. Here, we surveyed the expression patterns of commonly used Gal4 drivers in adult Drosophila female tissues under optimal conditions and found that multiple drivers have unreported secondary sites of expression beyond their published cell type/tissue expression pattern. These results underscore the importance of thoroughly characterizing Gal4 tools as part of a rigorous experimental design that avoids potential misinterpretation of results as we strive for understanding how the function of a specific gene/pathway in one tissue contributes to whole-body physiology.

RanGTP induces an effector gradient of XCTK2 and importin α/ß for spindle microtubule cross-linking.

High RanGTP around chromatin is important for governing spindle assembly during meiosis and mitosis by releasing the inhibitory effects of importin α/ß. Here we examine how the Ran gradient regulates Kinesin-14 function to control spindle organization. We show that Xenopus Kinesin-14, XCTK2, and importin α/ß form an effector gradient that is highest at the poles and diminishes toward the chromatin, which is opposite the RanGTP gradient. Importin α and ß preferentially inhibit XCTK2 antiparallel microtubule cross-linking and sliding by decreasing the microtubule affinity of the XCTK2 tail domain. This change in microtubule affinity enables RanGTP to target endogenous XCTK2 to the spindle. We propose that these combined actions of the Ran pathway are critical to promote Kinesin-14 parallel microtubule cross-linking to help focus spindle poles for efficient bipolar spindle assembly. Furthermore, our work illustrates that RanGTP regulation in the spindle is not simply a switch, but rather generates effector gradients where importins α and ß gradually tune the activities of spindle assembly factors.

The nuclear receptor seven up functions in adipocytes and oenocytes to control distinct steps of Drosophila oogenesis.

Reproduction is intimately linked to the physiology of an organism. Nuclear receptors are widely expressed transcription factors that mediate the effects of many circulating molecules on physiology and reproduction. While multiple studies have focused on the roles of nuclear receptors intrinsically in the ovary, it remains largely unknown how the actions of nuclear receptors in peripheral tissues influence oogenesis. We identified the nuclear receptor encoded by svp as a novel regulator of oogenesis in adult Drosophila. Global somatic knockdown of svp reduces egg production by increasing GSC loss, death of early germline cysts, and degeneration of vitellogenic follicles. Tissue-specific knockdown experiments revealed that svp remotely controls these different steps of oogenesis through separate mechanisms involving distinct tissues. Specifically, adipocyte-specific svp knockdown impairs GSC maintenance and early germline cyst survival, whereas oenocyte-specific svp knockdown increases the death of vitellogenic follicles without any effects on GSCs or early cysts. These results illustrate that nuclear receptors can control reproduction through a variety of mechanisms involving peripheral tissues.

Maintenance of Proper Germline Stem Cell Number Requires Adipocyte Collagen in Adult Drosophila Females.

Stem cells reside in specialized niches and are regulated by a variety of physiological inputs. Adipocytes influence whole-body physiology and stem cell lineages; however, the molecular mechanisms linking adipocytes to stem cells are poorly understood. Here, we report that collagen IV produced in adipocytes is transported to the ovary to maintain proper germline stem cell (GSC) number in adult Drosophila females. Adipocyte-derived collagen IV acts through ß-integrin signaling to maintain normal levels of E-cadherin at the niche, thereby ensuring proper adhesion to GSCs. These findings demonstrate that extracellular matrix components produced in adipocytes can be transported to and incorporated into an established adult tissue to influence stem cell number.

Gut healing: haemocytes aid via Sax and Tkv jazzes it down.

Control of stem cell activity is essential for accurate regeneration. Pathogen- or chemical-induced intestinal damage is now shown to recruit haemocytes expressing bone morphogenetic protein signals that stimulate proliferation of intestinal stem cells and subsequently induce their quiescence, in conjunction with muscle-derived bone morphogenetic proteins. A temporal switch in expression of Type I receptors enables this two-phase response.

The Ran-GTP gradient spatially regulates XCTK2 in the spindle.

Ran is a small GTP binding protein that was originally identified as a regulator of nucleocytoplasmic transport [1] and subsequently found to be important for spindle formation [2-5]. In mitosis, a gradient of Ran-GTP emanates from chromatin and diminishes toward spindle poles [6]. Ran-GTP promotes spindle self-organization through the release of importin-bound spindle assembly factors (SAFs), which stimulate microtubule (MT) nucleation and organization and regulate MT dynamics [7-9]. Although many SAFs are non-motile MT-associated proteins, such as NuMA, TPX2, and HURP [7, 10-12], Ran also controls motor proteins, including Kid and HSET/XCTK2 [13, 14]. The Kinesin-14 XCKT2 is important for spindle assembly and pole organization [15-20], and Ran-GTP is proposed to promote XCKT2 MT crosslinking activity by releasing importin α/ß from a bipartite nuclear localization signal (NLS) located in the tail domain [14]. Here, we show that the Ran-GTP gradient spatially regulates XCTK2 within the spindle. A flattened Ran-GTP gradient blocked the ability of excess XCTK2 to stimulate bipolar spindle assembly and resulted in XCTK2-mediated bundling of free MTs. These effects required the XCTK2 tail, which promoted the motility of XCTK2 within the spindle independent of the Ran-GTP gradient. In addition, the turnover kinetics of XCTK2 were spatially controlled: they were faster near the poles relative to the chromatin, but not with a mutant XCTK2 that cannot bind to importin α/ß. Our results support a model in which the Ran-GTP gradient spatially coordinates motor localization with motility to ensure efficient spindle formation.

Spatial gradients controlling spindle assembly.

The mitotic spindle is the macromolecular machine utilized to accurately segregate chromosomes in cells. How this self-organized structure assembles is a key aspect of understanding spindle morphogenesis. In the present review, we focus on understanding mechanisms of spindle self-assembly and address how subcellular signalling gradients, such as Ran-GTP and Aurora B, contribute to spindle organization and function.

Kif18A uses a microtubule binding site in the tail for plus-end localization and spindle length regulation.

The mitotic spindle is a macromolecular structure utilized to properly align and segregate sister chromatids to two daughter cells. During mitosis, the spindle maintains a constant length, even though the spindle microtubules (MTs) are constantly undergoing polymerization and depolymerization [1]. Members of the kinesin-8 family are important for the regulation of spindle length and for chromosome positioning [2-9]. Kinesin-8 proteins are length-specific, plus-end-directed motors that are proposed to be either MT depolymerases [3, 4, 8, 10, 11] or MT capping proteins [12]. How Kif18A uses its destabilization activity to control spindle morphology is not known. We found that Kif18A controls spindle length independently of its role in chromosome positioning. The ability of Kif18A to control spindle length is mediated by an ATP-independent MT binding site at the C-terminal end of the Kif18A tail that has a strong affinity for MTs in vitro and in cells. We used computational modeling to ask how modulating the motility or binding properties of Kif18A would affect its activity. Our modeling predicts that both fast motility and a low off rate from the MT end are important for Kif18A function. In addition, our studies provide new insight into how depolymerizing and capping enzymes can lead to MT destabilization.

Kinesin-8s hang on by a tail.

Accurate segregation of genetic material into two daughter cells is essential for organism reproduction, development, and survival. The cell assembles a macromolecular structure called the mitotic spindle, which is composed of dynamic microtubules (MTs) and many associated proteins that assemble the spindle and drive the segregation of the chromosomes. Members of the kinesin superfamily of MT associated proteins use the energy of ATP hydrolysis to help organize the spindle, to transport cargo within the spindle, and to regulate spindle MT dynamics. The Kinesin-8 and Kinesin-13 families are involved in controlling mitotic spindle morphology, spindle positioning, and chromosome movement. While both kinesin families are MT destabilizing enzymes, it is unclear whether their mechanisms of MT destabilization are mechanistically similar or how they act to destabilize MTs. Recently, three groups identified an additional MT binding domain within the tail of Kinesin-8s that is essential for their roles in regulating MT dynamics and chromosome positioning.

Proper organization of microtubule minus ends is needed for midzone stability and cytokinesis.

Successful cytokinesis is critical for maintaining genome stability and requires the assembly of a robust central spindle to specify the cleavage furrow position, to prevent separated chromosomes from coming back together, and to contribute to midbody abscission. A proper central spindle is assembled and maintained by a number of microtubule-associated and molecular motor proteins that sort microtubules into bundles with their plus ends overlapping at the center. The mechanisms by which different factors organize the central spindle microtubules remain unclear. We found that perturbation of the minus-end-directed Kinesin-14 HSET increased the frequency of polyploid cells, which resulted from a failure in cytokinesis. In addition, HSET knockdown resulted in severe midzone microtubule organization, most notably at microtubule minus ends, as well as mislocalization of several midbody-associated proteins. Biochemical analysis showed that both human HSET and Xenopus XCTK2 cofractionated with the gamma-tubulin ring complexes on sucrose gradients and that XCTK2 associated with gamma-tubulin and Xgrip109 by immunoprecipitation. Our data reveal the novel finding that a minus-end-directed motor contributes to the organization and stability of the central spindle, which is needed for proper cytokinesis.

Kinesin-14 family proteins HSET/XCTK2 control spindle length by cross-linking and sliding microtubules.

Kinesin-14 family proteins are minus-end directed motors that cross-link microtubules and play key roles during spindle assembly. We showed previously that the Xenopus Kinesin-14 XCTK2 is regulated by Ran via the association of a bipartite NLS in the tail of XCTK2 with importin alpha/beta, which regulates its ability to cross-link microtubules during spindle formation. Here we show that mutation of the nuclear localization signal (NLS) of human Kinesin-14 HSET caused an accumulation of HSET in the cytoplasm, which resulted in strong microtubule bundling. HSET overexpression in HeLa cells resulted in longer spindles, similar to what was seen with NLS mutants of XCTK2 in extracts, suggesting that Kinesin-14 proteins play similar roles in extracts and in somatic cells. Conversely, HSET knockdown by RNAi resulted in shorter spindles but did not affect pole formation. The change in spindle length was not dependent on K-fibers, as elimination of the K-fiber by Nuf2 RNAi resulted in an increase in spindle length that was partially rescued by co-RNAi of HSET. However, these changes in spindle length did require microtubule sliding, as overexpression of an HSET mutant that had its sliding activity uncoupled from its ATPase activity resulted in cells with spindle lengths shorter than cells overexpressing wild-type HSET. Our results are consistent with a model in which Ran regulates the association of Kinesin-14s with importin alpha/beta to prevent aberrant cross-linking and bundling of microtubules by sequestering Kinesin-14s in the nucleus during interphase. Kinesin-14s act during mitosis to cross-link and slide between parallel microtubules to regulate spindle length.