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Diet-induced obesity leads to dysfunctional feeding behavior. However, the precise molecular nodes underlying diet-induced feeding motivation dysregulation are poorly understood. The fruit fly is a simple genetic model system yet displays significant evolutionary conservation to mammalian nutrient sensing and energy balance. Using a longitudinal high-sugar regime in Drosophila, we sought to address how diet-induced changes in adipocyte lipid composition regulate feeding behavior. We observed that subjecting adult Drosophila to a prolonged high-sugar diet degrades the hunger-driven feeding response. Lipidomics analysis reveals that longitudinal exposure to high-sugar diets significantly alters whole-body phospholipid profiles. By performing a systematic genetic screen for phospholipid enzymes in adult fly adipocytes, we identify Pect as a critical regulator of hunger-driven feeding. Pect is a rate-limiting enzyme in the phosphatidylethanolamine (PE) biosynthesis pathway and the fly ortholog of human PCYT2. We show that disrupting Pect activity only in the Drosophila fat cells causes insulin resistance, dysregulated lipoprotein delivery to the brain, and a loss of hunger-driven feeding. Previously human studies have noted a correlation between PCYT2/Pect levels and clinical obesity. Now, our unbiased studies in Drosophila provide causative evidence for adipocyte Pect function in metabolic homeostasis. Altogether, we have uncovered that PE phospholipid homeostasis regulates hunger response.

Evolutionary transitions are frequently associated with novel anatomical structures

Diet-induced obesity (DIO) leads to dysfunctional feeding behavior. However, the precise molecular nodes underlying diet-induced dysregulation of satiety sensing and feeding motivation are poorly understood. The fruit fly is a simple genetic model system yet displays significant evolutionary conservation to mammalian nutrient sensing and energy balance. Using a longitudinal high sugar regime in Drosophila, we sought to address how lipid alteration in fat cells alters feeding behavior. We find that prolonged exposure to HSD degrades the hunger-driven feeding (HDF) response. Lipidomics analysis reveals that longitudinal exposure to HSD significantly alters whole body phospholipid profiles. By performing a systematic screen for phospholipid enzymes, we identify Pect as a critical regulator of hunger-driven feeding. Pect is a rate-limiting enzyme in the phosphatidylethanolamine (PE) biosynthesis pathway and the fly ortholog of human PCYT2. We show that disrupting Pect only in the fat body causes insulin-resistant phenotypes and a loss of hunger-driven feeding. Excitingly, we find that overexpression of Pect restores HSD-induced loss of hunger-driven feeding response. Strikingly human studies have noted a correlation between PCYT2/Pect levels and clinical obesity. Now, our unbiased studies in Drosophila provide specific genetic evidence for Pect in maintaining nutrient sensing during DIO. Our study provides novel insights into the role of phospholipids in interorgan communication of nutrient status.

SummaryEnergy sensing neural circuits decide to expend or conserve resources by integrating tonic steady-state energy store information with phasic signals for hunger and food intake. Tonic signals, in the form of adipose tissue-derived adipokines, set the baseline level of energy-sensing neuron activity, providing context for interpretation of phasic messages. However, the mechanism by which tonic adipokine information establishes baseline neuronal function is unclear. Here we show that Upd2, a Drosophila Leptin ortholog, regulates actin-based synapse reorganization by reducing inhibitory synaptic contacts, thereby providing a permissive neural tone for insulin release under conditions of nutrient surplus. Unexpectedly, Insulin acts on the same upstream inhibitory neurons to conversely increase synapse number, hence re-instating negative tone. Our results suggest that two surplus-sensing hormonal systems, Leptin/Upd2 and Insulin, converge on a neuronal circuit with opposing outcomes that establish tonic, energy-store-dependent neuron activity.HighlightsThe adipokine Upd2 regulates number of inhibitory synaptic contacts on Insulin neurons.Upd2 activates an actin-regulating complex of Arouser, Basigin, and Gelsolin in target neurons.Arouser, Basigin, and Gelsolin reduce the extent of inhibitory contact on Insulin neurons.Insulin resets negative tone by increasing the number of synaptic contacts made by its own upstream inhibitory neurons.

Summary Energy-sensing neural circuits decide to expend or conserve resources based, in part, on the tonic, steady-state, energy-store information they receive. Tonic signals, in the form of adipose tissue-derived adipokines, set the baseline level of activity in the energy-sensing neurons, thereby providing context for interpretation of additional inputs. However, the mechanism by which tonic adipokine information establishes steady-state neuronal function has heretofore been unclear. We show here that under conditions of nutrient surplus, Upd2, a Drosophila leptin ortholog, regulates actin-based synapse reorganization to reduce bouton number in an inhibitory circuit, thus establishing a neural tone that is permissive for insulin release. Unexpectedly, we found that insulin feeds back on these same inhibitory neurons to conversely increase bouton number, resulting in maintenance of negative tone. Our results point to a mechanism by which two surplus-sensing hormonal systems, Upd2/leptin and insulin, converge on a neuronal circuit with opposing outcomes to establish energy-store-dependent neuron activity.

Background: Vertebrate somites are subdivided into lineage compartments, each with distinct cell fates and evolutionary histories. Insights into somite evolution can come from studying amphioxus, the best extant approximation of the chordate ancestor. Amphioxus somites have myotome and non-myotome compartments, but development and fates of the latter are incompletely described. Further, while epithelial to mesenchymal transition (EMT) is important for most vertebrate somitic lineages, amphioxus somites generally have been thought to remain entirely epithelial. Here, we examined amphioxus somites and derivatives, as well as extracellular matrix of the axial support system, in a series of developmental stages by transmission electron microscopy (TEM) and in situ hybridization for collagen expression. Results: The amphioxus somite differentiates medially into myotome, laterally into the external cell layer (a sub-dermal mesothelium), ventrally into a bud that forms mesothelia of the perivisceral coelom, and ventro-medially into the sclerotome. The sclerotome forms initially as a monolayered cell sheet that migrates between the myotome and the notochord and neural tube; subsequently, this cell sheet becomes double layered and encloses the sclerocoel. Other late developments include formation of the fin box mesothelia from lateral somites and the advent of isolated fibroblasts, likely somite derived, along the myosepta. Throughout development, all cells originating from the non-myotome regions of somites strongly express a fibrillar collagen gene, ColA, and thus likely contribute to extracellular matrix of the dermal and axial connective tissue system. Conclusions: We provide a revised model for the development of amphioxus sclerotome and fin boxes and confirm previous reports of development of the myotome and lateral somite. In addition, while somite derivatives remain almost entirely epithelial, limited de-epithelialization likely converts some somitic cells into fibroblasts of the myosepta and dermis. Ultrastructure and collagen expression suggest that all non-myotome somite derivatives contribute to extracellular matrix of the dermal and axial support systems. Although amphioxus sclerotome lacks vertebrate-like EMT, it resembles that of vertebrates in position, movement to surround midline structures and into myosepta, and contribution to extracellular matrix of the axial support system. Thus, many aspects of the sclerotome developmental program evolved prior to the origin of the vertebrate mineralized skeleton. Keywords: Somite evolution Sclerotome Skeleton Connective tissue Tendon Amphioxus Chordate

One of the earliest steps of embryonic development is the establishment of polarity along the anteroposterior axis. Extensive studies of Drosophila embryonic development have elucidated mechanisms for establishing polarity, while studies with other model systems have found that many of these molecular components are conserved through evolution. One exception is Bicoid, the master organizer of anterior development in Drosophila and higher dipterans, which is not conserved. Thus, the study of anteroposterior patterning in insects that lack Bicoid can provide insight into the evolution of the diversity of body plan patterning networks. To this end, we have established the long germ parasitic wasp Nasonia vitripennis as a model for comparative studies with Drosophila.Here we report that, in Nasonia, a gradient of localized caudal mRNA directs posterior patterning, whereas, in Drosophila, the gradient of maternal Caudal protein is established through translational repression by Bicoid of homogeneous caudalmRNA. Loss of caudal function in Nasonia results in severe segmentation defects. We show that Nasonia caudal is an activator of gap gene expression that acts far towards the anterior of the embryo, placing it atop a cascade of early patterning. By contrast, activation of gap genes in flies relies on redundant functions of Bicoid and Caudal, leading to a lack of dramatic action on gap gene expression: caudal instead plays a limited role as an activator of pair-rule gene expression. These studies,together with studies in short germ insects, suggest that caudal is an ancestral master organizer of patterning, and that its role has been reduced in higher dipterans such as Drosophila.

Proper formation of the musculoskeletal system requires the coordinated development of the muscle, cartilage and tendon lineages arising from the somitic mesoderm. During early somite development, muscle and cartilage emerge from two distinct compartments, the myotome and sclerotome, in response to signals secreted from surrounding tissues. As the somite matures, the tendon lineage is established within the dorsolateral sclerotome, adjacent to and beneath the myotome. We examine interactions between the three lineages by observing tendon development in mouse mutants with genetically disrupted muscle or cartilage development. Through analysis of embryos carrying null mutations in Myf5 and Myod1, hence lacking both muscle progenitors and differentiated muscle, we identify an essential role for the specified myotome in axial tendon development, and suggest that absence of tendon formation in Myf5/Myod1 mutants results from loss of the myotomal FGF proteins, which depend upon Myf5 and Myod1 for their expression, and are required, in turn, for induction of the tendon progenitor markers. Our analysis of Sox5/Sox6 double mutants, in which the chondroprogenitors are unable to differentiate into cartilage,reveals that the two cell fates arising from the sclerotome, axial tendon and cartilage are alternative lineages, and that cartilage differentiation is required to actively repress tendon development in the dorsolateral sclerotome.

Embryonic anterior–posterior patterning is well understood in Drosophila, which uses ‘long germ’ embryogenesis, in which all segments are patterned before cellularization. In contrast, most insects use ‘short germ’ embryogenesis, wherein only head and thorax are patterned in a syncytial environment while the remainder of the embryo is generated after cellularization. We use the wasp Nasonia (Nv) to address how the transition from short to long germ embryogenesis occurred. Maternal and gap gene expression in Nasonia suggest long germ embryogenesis. However, the Nasonia pair-rule genes even-skipped, odd-skipped, runt and hairy are all expressed as early blastoderm pair-rule stripes and late-forming posterior stripes. Knockdown of Nv eve, odd or h causes loss of alternate segments at the anterior and complete loss of abdominal segments. We propose that Nasonia uses a mixed mode of segmentation wherein pair-rule genes pattern the embryo in a manner resembling Drosophila at the anterior and ancestral Tribolium at the posterior. DOI: http://dx.doi.org/10.7554/eLife.01440.001, eLife digest Networks of genes that work together are widespread in nature. The conservation of individual genes across species and the tendency of their networks to stick together is a sign that they are working efficiently. Furthermore, it is common for existing gene networks to be adapted to perform new tasks, instead of new networks being invented every time a similar but distinct demand arises. One important question is: how can evolution use the same building blocks—such as the genes in a functioning network—in different ways to achieve new outcomes? The gene network that sets up the ‘body plan’ of insects during development has been well studied, most deeply in the fruit fly, Drosophila. Like all insects, the body of a fruit fly is divided into three main parts—the head, the thorax and the abdomen—and each of these parts is made up of several smaller segments. There is a remarkable diversity of insect body plans in nature, and yet, these seem to arise from the same gene networks in the embryo. When a Drosophila embryo is growing into a larva, all the different body segments develop at the same time. In most other insects, however, segments of the abdomen emerge later and sequentially during the development process. The ancestors of most insects are also thought to have developed in this way, which is known as ‘short germ embryogenesis’. So how did the so-called ‘long germ embryogenesis’, as observed in Drosophila, evolve from the short germ embryogenesis that is observed in most other insects? The gene network that controls development includes the ‘pair-rule genes’ that are expressed in a pattern of alternating stripes that wrap around, top to bottom, along most of the length of the embryo. These stripes mark where the edges of each body segment will eventually develop. In fruit flies, this pattern extends along the entire length of the embryo and the stripes all appear at one time. However, in the abdominal region of short germ insects, the pair-rule genes are expressed in waves that pass through the posterior region as it grows, with new segments being added one behind the other. Now, Rosenberg et al. have attempted to explain how the same genes can be used to direct the segmentation process in such different ways by studying another long germ insect species, the jewel wasp. Analysis of the expression of pair-rule genes in the jewel wasp shows that it uses a mixed strategy to control segmentation. The development of segments at the front of its body is directed in the same way as the fruit fly, with all these segments laid down together. However, the segments at the rear of the body are only patterned later, one after the other, like most other insects. The work of Rosenberg et al. suggests that the jewel wasp represents an intermediate step between ancestral insects and Drosophila in the evolution of the gene network that patterns the ‘body plan’. Identifying and studying these intermediate forms allows us to understand the ways in which evolution can innovate by building upon what has come before. DOI: http://dx.doi.org/10.7554/eLife.01440.002

Somites are the bilaterally symmetric embryonic precursors of the vertebrate skeleton and axial muscle. Three recent studies reveal that somites form asymmetrically in the absence of retinoic acid signaling. These results uncover an unexpected relationship between somitogenesis and left-right patterning, and suggest that bilateral somite formation is regulated along the left-right axis.

In zebrafish, slow twitch muscle is specified from a somitic muscle precursor pool by local Hedgehog (Hh) signals. A new study identifies the transcription factor Blimp-1 as a key downstream mediator in this process.

Defects in tendon patterning and differentiation are seldom assessed in mouse mutants due to the difficulty in visualizing connective tissue structures. To facilitate tendon analysis, we have generated mouse lines harboring two different transgene reporters, alkaline phosphatase (AP) and green fluorescent protein (GFP), each expressed using regulatory elements derived from the endogenous Scleraxis (Scx) locus. Scx encodes a transcription factor expressed in all developing tendons and ligaments as well as in their progenitors. Both the ScxGFP and ScxAP transgenes are expressed in patterns recapitulating almost entirely the endogenous developmental expression of Scx including very robust expression in the tendons and ligaments. These reporter lines will facilitate isolation of tendon cells and phenotypic analysis of these tissues in a variety of genetic backgrounds.

The Bicoid (Bcd) gradient in Drosophila has long been a model for the action of a morphogen in establishing embryonic polarity. However, it is now clear that bcd is a unique feature of higher Diptera. An evolutionarily ancient gene, orthodenticle (otd), has a bcd-like role in the beetle Tribolium. Unlike the Bcd gradient, which arises by diffusion of protein from an anteriorly localized messenger RNA, the Tribolium Otd gradient forms by translational repression of otd mRNA by a posteriorly localized factor. These differences in gradient formation are correlated with differences in modes of embryonic patterning. Drosophila uses long germ embryogenesis, where the embryo derives from the entire anterior-posterior axis, and all segments are patterned at the blastoderm stage, before gastrulation. In contrast, Tribolium undergoes short germ embryogenesis: the embryo arises from cells in the posterior of the egg, and only anterior segments are patterned at the blastoderm stage, with the remaining segments arising after gastrulation from a growth zone. Here we describe the role of otd in the long germband embryo of the wasp Nasonia vitripennis. We show that Nasonia otd maternal mRNA is localized at both poles of the embryo, and resulting protein gradients pattern both poles. Thus, localized Nasonia otd has two major roles that allow long germ development. It activates anterior targets at the anterior of the egg in a manner reminiscent of the Bcd gradient, and it is required for pre-gastrulation expression of posterior gap genes.

Recent research has broadened significantly our understanding of how the somite, a specialized mesodermal structure found in vertebrate embryos, gives rise to the cartilage, muscle and tendon cell lineages. The specification of somite derivatives involves the action of patterning signals secreted from adjacent tissue combined with the activation, in particular somitic compartments, of genes promoting cell lineage specification.

Little is known about the genesis and patterning of tendons and other connective tissues, mostly owing to the absence of early markers. We have found that Scleraxis, a bHLH transcription factor, is a highly specific marker for all the connective tissues that mediate attachment of muscle to bone in chick and mouse, including the limb tendons, and show that early scleraxis expression marks the progenitor cell populations for these tissues. In the early limb bud, the tendon progenitor population is found in the superficial proximomedial mesenchyme. Using the scleraxis gene as a marker we show that these progenitors are induced by ectodermal signals and restricted by bone morphogenetic protein (BMP) signaling within the mesenchyme. Application of Noggin protein antagonizes this endogenous BMP activity and induces ectopic scleraxis expression. However, the presence of excess tendon progenitors does not lead to the production of additional or longer tendons, indicating that additional signals are required for the final formation of a tendon. Finally, we show that the endogenous expression of noggin within the condensing digit cartilage contributes to the induction of distal tendons.