Andrew P. Bailey

Multi-isotope imaging mass spectrometry quantifies stem cell division and metabolism

Mass spectrometry with stable isotope labels has been seminal in discovering the dynamic state of living matter1,2 but is limited to bulk tissues or cells. We developed multi-isotope imaging mass spectrometry (MIMS) that allowed us to view and measure stable isotope incorporation with sub-micron resolution3,4. Here we apply MIMS to diverse organisms, including Drosophila, mice, and humans. We test the “immortal strand hypothesis,” which predicts that during asymmetric stem cell division chromosomes containing older template DNA are segregated to the daughter destined to remain a stem cell, thus insuring lifetime genetic stability. After labeling mice with 15N-thymidine from gestation through post-natal week 8, we find no 15N label retention by dividing small intestinal crypt cells after 4wk chase. In adult mice administered 15N-thymidine pulse-chase, we find that proliferating crypt cells dilute label consistent with random strand segregation. We demonstrate the broad utility of MIMS with proof-of-principle studies of lipid turnover in Drosophila and translation to the human hematopoietic system. These studies show that MIMS provides high-resolution quantitation of stable isotope labels that cannot be obtained using other techniques and that is broadly applicable to biological and medical research.Mass spectrometry with stable isotope labels has been seminal in discovering the dynamic state of living matter, but is limited to bulk tissues or cells. We developed multi-isotope imaging mass spectrometry (MIMS) that allowed us to view and measure stable isotope incorporation with submicrometre resolution. Here we apply MIMS to diverse organisms, including Drosophila, mice and humans. We test the ‘immortal strand hypothesis’, which predicts that during asymmetric stem cell division chromosomes containing older template DNA are segregated to the daughter destined to remain a stem cell, thus insuring lifetime genetic stability. After labelling mice with 15N-thymidine from gestation until post-natal week 8, we find no 15N label retention by dividing small intestinal crypt cells after a four-week chase. In adult mice administered 15N-thymidine pulse-chase, we find that proliferating crypt cells dilute the 15N label, consistent with random strand segregation. We demonstrate the broad utility of MIMS with proof-of-principle studies of lipid turnover in Drosophila and translation to the human haematopoietic system. These studies show that MIMS provides high-resolution quantification of stable isotope labels that cannot be obtained using other techniques and that is broadly applicable to biological and medical research.

Enteric Neurons and Systemic Signals Couple Nutritional and Reproductive Status with Intestinal Homeostasis

Summary The gastrointestinal tract is emerging as a key regulator of appetite and metabolism, but daunting neuroanatomical complexity has hampered identification of the relevant signals. Invertebrate models could provide a simple and genetically amenable alternative, but their autonomic nervous system and its visceral functions remain largely unexplored. Here we develop a quantitative method based on defecation behavior to uncover a central role for the Drosophila intestine in the regulation of nutrient intake, fluid, and ion balance. We then identify a key homeostatic role for autonomic neurons and hormones, including a brain-gut circuit of insulin-producing neurons modulating appetite, a vasopressin-like system essential for fluid homeostasis, and enteric neurons mediating sex peptide-induced changes in intestinal physiology. These conserved mechanisms of visceral control, analogous to those found in the enteric nervous system and hypothalamic/pituitary axis, enable the study of autonomic control in a model organism that has proved instrumental in understanding sensory and motor systems.

Antioxidant Role for Lipid Droplets in a Stem Cell Niche of Drosophila.

Summary Stem cells reside in specialized microenvironments known as niches. During Drosophila development, glial cells provide a niche that sustains the proliferation of neural stem cells (neuroblasts) during starvation. We now find that the glial cell niche also preserves neuroblast proliferation under conditions of hypoxia and oxidative stress. Lipid droplets that form in niche glia during oxidative stress limit the levels of reactive oxygen species (ROS) and inhibit the oxidation of polyunsaturated fatty acids (PUFAs). These droplets protect glia and also neuroblasts from peroxidation chain reactions that can damage many types of macromolecules. The underlying antioxidant mechanism involves diverting PUFAs, including diet-derived linoleic acid, away from membranes to the core of lipid droplets, where they are less vulnerable to peroxidation. This study reveals an antioxidant role for lipid droplets that could be relevant in many different biological contexts.

Volume Determination with Two Standards Allows Absolute Quantification and Improved Chemometric Analysis of Metabolites by NMR from Submicroliter Samples

The accurate measurement of metabolite concentrations in miniscule biological sample volumes is often desirable, yet it remains challenging. In many cases, the starting analyte volumes are imprecisely known, or not directly measurable, and hence absolute metabolite concentrations are difficult to calculate. Here, we introduce volume determination using two standards (VDTS) as a general quantitative method for the analysis of polar metabolites in submicrolitre samples using 1H NMR spectroscopy. This approach permits the back calculation of absolute metabolite concentrations from small biological samples of unknown volume. Where small sample volumes are also variable, VDTS can improve multivariate chemometric analysis. In this context, principal component analysis (PCA) yielded more logically consistent and biologically insightful outputs when we used volume-corrected spectra, calculated using VDTS, rather than probabilistic quotient normalization (PQN) of raw spectra. As proof-of-principle, the VDTS-based method and PCA were used to analyze polar metabolites in the hemolymph (blood) extracted from larvae of the very small but widely used genetic model organism Drosophila. This analysis showed that the hemolymph metabolomes of males and females are markedly different when larvae are well fed. However, gender-specific metabolomes tend to converge when larval dietary nutrients are restricted. We discuss the biological implications of these surprising results and compare and contrast them to previous analyses of Drosophila hemolymph and mammalian blood plasma. Together, these findings reveal an interesting and hitherto unknown sexual dimorphism in systemic Drosophila metabolites, clearly warranting further biological investigation. Importantly, the VDTS approach should be adaptable to many different analytical platforms, including mass spectrometry.

Stable isotope analysis of dynamic lipidomics

Metabolic pathway flux is a fundamental element of biological activity, which can be quantified using a variety of mass spectrometric techniques to monitor incorporation of stable isotope-labelled substrates into metabolic products. This article contrasts developments in electrospray ionisation mass spectrometry (ESI-MS) for the measurement of lipid metabolism with more established gas chromatography mass spectrometry and isotope ratio mass spectrometry methodologies. ESI-MS combined with diagnostic tandem MS/MS scans permits the sensitive and specific analysis of stable isotope-labelled substrates into intact lipid molecular species without the requirement for lipid hydrolysis and derivatisation. Such dynamic lipidomic methodologies using non-toxic stable isotopes can be readily applied to quantify lipid metabolic fluxes in clinical and metabolic studies in vivo. However, a significant current limitation is the absence of appropriate software to generate kinetic models of substrate incorporation into multiple products in the time domain. Finally, we discuss the future potential of stable isotope-mass spectrometry imaging to quantify the location as well as the extent of lipid synthesis. This article is part of a Special Issue entitled: BBALIP_Lipidomics Opinion Articles edited by Sepp Kohlwein.

PS4.59Developmental diet impacts on Drosophila lifespan via lipid autotoxins

It has recently been shown that tail regeneration in Xenopus tadpoles require a sustained increase in the production of reactive oxygen species (ROS). We have also found that embryos are associated with sustained high levels of ROS, which are also needed for tissue development to proceed. It is not entirely clear what is the source of ROS during embryogenesis and following injury, and whether it is identical for both processes. Furthermore, it is unclear what specific type(s) of ROS are required for either process. One of the primary aims of this project is to identify the source of ROS and their nature, during embryogenesis and following injury. In both cases, particular focus is made on hydrogen peroxide (H2O2) and its precursor superoxide (O2-). A second primary aim is to determine the downstream targets of O2and H2O2, particularly during tissue formation, repair and regeneration. To address these aims a model with high regenerative capacity has been chosen, namely frog embryos and tadpoles. Characterisation of specific ROS involved is made possible through the use of the electron paramagnetic resonance (EPR) spectroscopy and fluorescent imaging. Ultimately, we are investigating: to what extent developmental processes are reactivated during regeneration, in the context of ROS; and whether manipulating specific ROS levels might provide a mechanism by which we may induce and sustain a better, more complete regenerative response in humans, following injury.

02-P005 A mechanism for “brain sparing” during dietary restriction in Drosophila

phology. We have generated a Tg(phd3:GFP)/+; vhl1/+ transgenic line, which exhibit robust GFP expression in the vhl1/vhl1 background, essentially acting as an in vivo fluorescent reporter to identify the mutant cells/embryos. To induce VHL kidney tumors in fish, we have utilized this transgenic line to generate donor embryos and transplanted the mutant blastomeres to the wild type host embryos, specifically targeting these mutant cells to the renal primordium. We report that Tg(phd3:GFP) line is a live reporter for monitoring hypoxia and the induction of HIF signaling. We shall present our observations on the fate of the vhl1 mutant cells in the chimeric fish and also our analysis for the possible occurrence of neoplasia in these chimeric fish.

12-P004 Development and function of visceral innervation in Drosophila

and ectopic expression, we demonstrated that Gata2 regulates GABAergic neuron development in the midbrain, but not in the rhombomere1. Without Gata2, all the precursors in the embryonic midbrain fail to activate GABAergic neuron-specific gene expression and switch to a glutamatergic phenotype instead. Surprisingly, this fate switch is also observed throughout the neonatal midbrain, except for the GABAergic neurons located in the ventral dopaminergic nuclei, suggesting a distinct developmental pathway for these neurons. We have further investigated the origin, developmental history and regulatory mechanisms of these GABAergic neurons associated with the ventral dopaminergic nuclei. The presented studies identify Gata2 as an essential postmitotic selector of the GABAergic neurotransmitter identity and demonstrate developmental heterogeneity of the GABAergic neurons in the midbrain.