Improved whole-mount immunofluorescence protocol for consistent and robust labeling of adult Drosophila melanogaster adipose tissue.

Energy storage and endocrine functions of the Drosophila fat body make it an excellent model for elucidating mechanisms that underlie physiological and pathophysiological organismal metabolism. Combined with Drosophila's robust genetic and immunofluorescence microscopy toolkits, studies of Drosophila fat body function are ripe for cell biological analysis. Unlike the larval fat body, which is easily removed as a single, cohesive sheet of tissue, isolating intact adult fat body proves to be more challenging, thus hindering consistent immunofluorescence labeling even within a single piece of adipose tissue. Here, we describe an improved approach to handling Drosophila abdomens that ensures full access of the adult fat body to solutions generally used in immunofluorescence labeling protocols. In addition, we assess the quality of fluorescence reporter expression and antibody immunoreactivity in response to variations in fixative type, fixation incubation time, and detergent used for cellular permeabilization. Overall, we provide several recommendations for steps in a whole-mount staining protocol that results in consistent and robust immunofluorescence labeling of the adult Drosophila fat body.

Host-derived Interleukin 1α induces an immunosuppressive tumor microenvironment via regulating monocyte-to-macrophage differentiation.

Tumor-associated macrophages exhibit high heterogeneity and contribute to the establishment of an immunosuppressive tumor microenvironment (TME). Although numerous studies have demonstrated that extracellular factors promote macrophage proliferation and polarization, the regulatory mechanisms governing the differentiation process to generate phenotypically, and functionally diverse macrophage subpopulations remain largely unexplored. In this study, we examined the influence of interleukin 1α (IL-1α) on the development of an immunosuppressive TME using orthotopic transplantation murine models of breast cancer. Deletion of host Il1α led to the rejection of inoculated congenic tumors. Single-cell sequencing analysis revealed that CX3CR1+ macrophage cells were the primary sources of IL-1α in the TME. The absence of IL-1α reprogrammed the monocyte-to-macrophage differentiation process within the TME, characterized by a notable decrease in the subset of CX3CR+ ductal-like macrophages and an increase in iNOS-expressing inflammatory cells. Comparative analysis of gene signatures in both human and mouse macrophage subsets suggested that IL-1α deficiency shifted the macrophage polarization from M2 to M1 phenotypes, leading to enhanced cytotoxic T lymphocyte activity in the TME. Importantly, elevated levels of IL-1α in human cancers were associated with worse prognosis following immunotherapy. These findings underscore the pivotal role of IL-1α in shaping an immune-suppressive TME through the regulation of macrophage differentiation and activity, highlighting IL-1α as a potential target for breast cancer treatment.

Differential amino acid transporter expression in adult Drosophila melanogaster tissues.

Organismal macronutrient intake modulates organ and tissue function. Dietary amino acids play essential roles in metabolic processes that support normal tissue growth, repair, and function. For example, in Drosophila melanogaster , protein-deficient diets lead to reduced overall organismal growth during larval development and severely decreased egg production in adult females. Multiple tissues, therefore, must sense and respond to dietary protein input. Amino acid transporter proteins facilitate the movement of amino acids across cellular membranes. Based on high-throughput expression studies, the Drosophila genome is predicted to encode 58 amino acid transporters. We have set out to determine if there are tissue-specific amino acid requirements for proper tissue function by first assessing the complement of amino acid transporters expressed in several adult tissues. Using RT-PCR to assess transcript levels, we find that most of the 24 amino acid transporters examined are expressed in the head, thorax, abdomen, gut, and ovary, while a subset shows differential transcript expression. This work will serve as the foundation for future studies addressing the impact of physiological factors, like nutrition, on amino acid sensing by individual tissues.

Ras/MAPK signaling mediates adipose tissue control of ovarian germline survival and ovulation in Drosophila melanogaster.

From insects to humans, oogenesis is tightly linked to nutritional input, yet little is known about how whole organism physiology matches dietary changes with oocyte development. Considering that diet-induced adipose tissue dysfunction is associated with an increased risk for fertility problems, and other obesity-associated pathophysiologies, it is critical to decipher the cellular and molecular mechanisms linking adipose nutrient sensing to remote control of the ovary and other tissues. Our previous studies in Drosophila melanogaster have shown that amino acid sensing, via the amino acid response pathway and mTOR-mediated signaling function within adipocytes to control germline stem cell maintenance and ovulation, respectively. Additionally, we demonstrated that insulin/insulin-like growth factor signaling within adipocytes employs distinct effector axes, PI3K/Akt1-dependent and -independent, downstream of insulin receptor activity to mediate fat-to-ovary communication. Here, we report that the Ras/MAPK signaling axis functions in adipocytes to regulate early germline cyst survival and ovulation of mature oocytes but is not important for germline stem cell maintenance or the progression through vitellogenesis. Thus, these studies uncover the complexity of signaling pathway activity that mediates inter-organ communication.

Methods to Analyze Nutritional and Inter-Organ Control of Drosophila Ovarian Germline Stem Cells.

Physiological status, particularly dietary input, has major impacts on the Drosophila melanogaster ovarian germline stem cell lineage. Moreover, several studies have shed light on the role that inter-organ communication plays in coordinating whole-organism responses to changes in physiology. For example, nutrient-sensing signaling pathways function within the fat body to regulate germline stem cells and their progeny in the ovary. Together with its incredible genetic and cell biological toolkits, Drosophila serves as an amenable model organism to use for uncovering molecular mechanisms that underlie physiological control of adult stem cells. In this methods chapter, we describe a general dietary manipulation paradigm, genetic manipulation of adult adipocytes, and whole-mount ovary immunofluorescence to investigate physiological control of germline stem cells.

How do DEI initiatives impact STEMM, and why do we still need them?

The number of diversity, equity, and inclusion (DEI) initiatives in science, technology, engineering, mathematics, and medicine (STEMM) have grown over the last few years. We asked several Black scientists what impact they have and why STEMM still needs them. They answer these questions and describe how DEI initiatives should evolve.

Antibody development to identify components of IIS and mTOR signaling pathways in lepidopteran species, a set of non-model insects.

Nutritional stress impacts many insect species that have differing reproductive strategies and life histories, yet it is unclear how nutrient-sensing signaling pathways mediate tissue-specific responses to changes in dietary input. In Drosophila melanogaster , insulin/insulin-like growth factor (IIS) and mTOR-mediated signaling within adipocytes regulates oogenesis. To facilitate comparative study of nutrient-sensing pathway activity in the fat body, we developed antibodies to assess IIS (anti-FOXO) and mTOR signaling (anti-TOR) across three nymphalid species (Lepidoptera). By optimizing whole-mount fat body immunostaining, we find FOXO nuclear enrichment in adult adipocytes, like that observed in Drosophila . Additionally, we show a previously uncharacterized TOR localization pattern in the fat body.

Lessons Learned in Implementing an Aging Research Training Program for Underrepresented Minority Students.

This brief report provides an overview of lessons learned through evaluation of the first five years of the NIA-funded South Carolina-Advancing Diversity in Aging Research (SC-ADAR) undergraduate program, whose goal is to increase the number of qualified underrepresented minority (URM) students who pursue scientific graduate studies in programs focusing on medicine, science, technology, engineering, and mathematics and aging. Partnering with five Historically Black Colleges and Universities in South Carolina, we implemented a research training approach that included two consecutive summers of research training in a University of South Carolina faculty laboratory, as part of a comprehensive 24-month research education program. In addition to the mentored research experience in a laboratory, students had coursework in the biology of aging and social gerontology, with additional workshops tailored to emergent student needs including basic academic skills development, work-life management skills, reflective social experiences, and enhanced support in the transition from undergraduate to graduate school. We provide an overview of lessons learned throughout the early program period, and a description of the iterative changes we made in the program in response to this learning, all of which have been incorporated into the existing SC-ADAR program.

Drosophila melanogaster as a model for nutrient regulation of ovarian function.

Observed in a wide variety of organism, from invertebrates to mammals, nutritional status modulates the energetically costly effort of producing female gametes. Despite this long-standing link between nutrition and ovarian function, relatively little is known about the cellular and molecular mechanisms that underlie how dietary components modulate egg production. Drosophila melanogaster, with its powerful and extensive genetic tools as well as its well-characterized ovarian response to diet, has proven to be instrumental in addressing this issue. This review covers what we currently know about the dietary control of oogenesis in Drosophila and the salient features of the fruit fly that make it a model for nutritional control of ovarian function.

Insulin signaling acts in adult adipocytes via GSK-3ß and independently of FOXO to control Drosophila female germline stem cell numbers.

Tissue-specific stem cells are tied to the nutritional and physiological environment of adult organisms. Adipocytes have key endocrine and nutrient-sensing roles and have emerged as major players in relaying dietary information to regulate other organs. For example, previous studies in Drosophila melanogaster revealed that amino acid sensing as well as diet-dependent metabolic pathways function in adipocytes to influence the maintenance of female germline stem cells (GSCs). How nutrient-sensing pathways acting within adipocytes influence adult stem cell lineages, however, is just beginning to be elucidated. Here, we report that insulin/insulin-like growth factor signaling in adipocytes promotes GSC maintenance, early germline cyst survival, and vitellogenesis. Further, adipocytes use distinct mechanisms downstream of insulin receptor activation to control these aspects of oogenesis, all of which are independent of FOXO. We find that GSC maintenance is modulated by Akt1 through GSK-3ß, early germline cyst survival is downstream of adipocyte Akt1 but independent of GSK-3ß, and vitellogenesis is regulated through an Akt1-independent pathway in adipocytes. These results indicate that, in addition to employing different types of nutrient sensing, adipocytes can use distinct axes of a single nutrient-sensing pathway to regulate multiple stages of the GSC lineage in the ovary.

Adipocyte Metabolic Pathways Regulated by Diet Control the Female Germline Stem Cell Lineage in Drosophila melanogaster.

Nutrients affect adult stem cells through complex mechanisms involving multiple organs. Adipocytes are highly sensitive to diet and have key metabolic roles, and obesity increases the risk for many cancers. How diet-regulated adipocyte metabolic pathways influence normal stem cell lineages, however, remains unclear. Drosophila melanogaster has highly conserved adipocyte metabolism and a well-characterized female germline stem cell (GSC) lineage response to diet. Here, we conducted an isobaric tags for relative and absolute quantification (iTRAQ) proteomic analysis to identify diet-regulated adipocyte metabolic pathways that control the female GSC lineage. On a rich (relative to poor) diet, adipocyte Hexokinase-C and metabolic enzymes involved in pyruvate/acetyl-CoA production are upregulated, promoting a shift of glucose metabolism toward macromolecule biosynthesis. Adipocyte-specific knockdown shows that these enzymes support early GSC progeny survival. Further, enzymes catalyzing fatty acid oxidation and phosphatidylethanolamine synthesis in adipocytes promote GSC maintenance, whereas lipid and iron transport from adipocytes controls vitellogenesis and GSC number, respectively. These results show a functional relationship between specific metabolic pathways in adipocytes and distinct processes in the GSC lineage, suggesting the adipocyte metabolism-stem cell link as an important area of investigation in other stem cell systems.

Adipocyte amino acid sensing controls adult germline stem cell number via the amino acid response pathway and independently of Target of Rapamycin signaling in Drosophila.

How adipocytes contribute to the physiological control of stem cells is a critical question towards understanding the link between obesity and multiple diseases, including cancers. Previous studies have revealed that adult stem cells are influenced by whole-body physiology through multiple diet-dependent factors. For example, nutrient-dependent pathways acting within the Drosophila ovary control the number and proliferation of germline stem cells (GSCs). The potential role of nutrient sensing by adipocytes in modulating stem cells in other organs, however, remains largely unexplored. Here, we report that amino acid sensing by adult adipocytes specifically modulates the maintenance of GSCs through a Target of Rapamycin-independent mechanism. Instead, reduced amino acid levels and the consequent increase in uncoupled tRNAs trigger activation of the GCN2-dependent amino acid response pathway within adipocytes, causing increased rates of GSC loss. These studies reveal a new step in adipocyte-stem cell crosstalk.

Frizzled3 is required for neurogenesis and target innervation during sympathetic nervous system development.

The sympathetic nervous system has served as an amenable model system to investigate molecular mechanisms underlying developmental processes in the nervous system. While much attention has been focused on neurotrophic factors controlling survival and connectivity of postmitotic sympathetic neurons, relatively little is known about signaling mechanisms regulating development of sympathetic neuroblasts. Here, we report that Frizzled3 (Fz3), a member of the Wnt receptor family, is essential for maintenance of dividing sympathetic neuroblasts. In Fz3(-/-) mice, sympathetic neuroblasts exhibit decreased proliferation and premature cell cycle exit. Fz3(-/-) sympathetic neuroblasts also undergo enhanced apoptosis, which could not be rescued by eliminating the proapoptotic factor, Bax. These deficits result in reduced generation of sympathetic neurons and pronounced decreases in the size of sympathetic chain ganglia. Furthermore, the axons of sympathetic neurons that persist in Fz3(-/-) ganglia are able to extend out of sympathetic ganglia toward distal targets, but fail to fully innervate final peripheral targets. The cell cycle exit, but not target innervation, defects in Fz3(-/-) mice are phenocopied in mice with conditional ablation of ß-catenin, a component of canonical Wnt signaling, in sympathetic precursors. Sympathetic ganglia and innervation of target tissues appeared normal in mice lacking a core planar cell polarity (PCP) component, Vangl2. Together, our results suggest distinct roles for Fz3 during sympathetic neuron development; Fz3 acts at early developmental stages to maintain a pool of dividing sympathetic precursors, likely via activation of ß-catenin, and Fz3 functions at later stages to promote innervation of final peripheral targets by postmitotic sympathetic neurons.