Atg8/LC3 controls systemic nutrient surplus signaling in flies and humans.

Organisms experience constant nutritional flux. Mechanisms at the interface of opposing nutritional states-scarcity and surplus-enable organismal energy homeostasis. Contingent on nutritional stores, adipocytes secrete adipokines, such as the fat hormone leptin, to signal nutrient status to the central brain. Increased leptin secretion underlies metabolic dysregulation during common obesity, but the molecular mechanisms regulating leptin secretion from human adipocytes are poorly understood. Here, we report that Atg8/LC3 family proteins, best known for their role in autophagy during nutrient scarcity, play an evolutionarily conserved role during nutrient surplus by promoting adipokine secretion. We show that in a well-fed state, Atg8/LC3 promotes the secretion of the Drosophila functional leptin ortholog unpaired 2 (Upd2) and leptin from human adipocytes. Proteomic analyses reveal that LC3 directs leptin to a secretory pathway in human cells. We identified LC3-dependent extracellular vesicle (EV) loading and secretion (LDELS) as a required step for leptin release, highlighting a unique secretory route adopted by leptin in human adipocytes. In Drosophila, mutations to Upd2's Atg8 interaction motif (AIM) result in constitutive adipokine retention. Atg8-mediated Upd2 retention alters lipid storage and hunger response and rewires the bulk organismal transcriptome in a manner conducive to starvation survival. Thus, Atg8/LC3's bidirectional role in nutrient sensing-conveying nutrient surplus and responding to nutrient deprivation-enables organisms to manage nutrient flux effectively. We posit that decoding how bidirectional molecular switches-such as Atg8/LC3-operate at the nexus of nutritional scarcity and surplus will inform therapeutic strategies to tackle chronic metabolic disorders.

Evolutionary assembly and disassembly of the mammalian sternum.

Evolutionary transitions are frequently associated with novel anatomical structures,1 but the origins of the structures themselves are often poorly known. We use developmental, genetic, and paleontological data to demonstrate that the therian sternum was assembled from pre-existing elements. Imaging of the perinatal mouse reveals two paired sternal elements, both composed primarily of cells with lateral plate mesoderm origin. Location, articulations, and development identify them as homologs of the interclavicle and the sternal bands of synapsid outgroups. The interclavicle, not previously recognized in therians,2 articulates with the clavicle and differs from the sternal bands in both embryonic HOX expression and pattern of skeletal maturation. The sternal bands articulate with the ribs in two styles, most clearly differentiated by their association with sternebrae. Evolutionary trait mapping indicates that the interclavicle and sternal bands were independent elements throughout most of synapsid history. The differentiation of rib articulation styles and the subdivision of the sternal bands into sternebrae were key innovations likely associated with transitions in locomotor and respiratory mechanics.3,4 Fusion of the interclavicle and the anterior sternal bands to form a presternum anterior to the first sternebra was a historically recent innovation unique to therians. Subsequent disassembly of the radically reduced sternum of mysticete cetaceans was element specific, reflecting the constraints that conserved developmental programs exert on composite structures.

Fat body phospholipid state dictates hunger-driven feeding behavior.

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.

Hoxa5 Activity Across the Lateral Somitic Frontier Regulates Development of the Mouse Sternum.

The skeletal system derives from multiple embryonic sources whose derivatives must develop in coordination to produce an integrated whole. In particular, interactions across the lateral somitic frontier, where derivatives of the somites and lateral plate mesoderm come into contact, are important for proper development. Many questions remain about genetic control of this coordination, and embryological information is incomplete for some structures that incorporate the frontier, including the sternum. Hox genes act in both tissues as regulators of skeletal pattern. Here, we used conditional deletion to characterize the tissue-specific contributions of Hoxa5 to skeletal patterning. We found that most aspects of the Hoxa5 skeletal phenotype are attributable to its activity in one or the other tissue, indicating largely additive roles. However, multiple roles are identified at the junction of the T1 ribs and the anterior portion of the sternum, or presternum. The embryology of the presternum has not been well described in mouse. We present a model for presternum development, and show that it arises from multiple, paired LPM-derived primordia. We show evidence that HOXA5 expression marks the embryonic precursor of a recently identified lateral presternum structure that is variably present in therians.

Insulin and Leptin/Upd2 Exert Opposing Influences on Synapse Number in Fat-Sensing Neurons.

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.

Development of somites and their derivatives in amphioxus, and implications for the evolution of vertebrate somites.

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.

Dual mode of embryonic development is highlighted by expression and function of Nasonia pair-rule genes.

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.

Generation of transgenic tendon reporters, ScxGFP and ScxAP, using regulatory elements of the scleraxis gene.

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.

Permissive and instructive anterior patterning rely on mRNA localization in the wasp embryo.

The long-germ mode of embryogenesis, in which segments arise simultaneously along the anteriorposterior axis, has evolved several times in different lineages of the holometabolous, or fully metamorphosing, insects. Drosophila's long-germ fate map is established largely by the activity of the dipteran-specific Bicoid (Bcd) morphogen gradient, which operates both instructively and permissively to accomplish anterior patterning. By contrast, all nondipteran long-germ insects must achieve anterior patterning independently of bcd. We show that bcd's permissive function is mimicked in the wasp by a maternal repression system in which anterior localization of the wasp ortholog of giant represses anterior expression of the trunk gap genes so that head and thorax can properly form.

A caudal mRNA gradient controls posterior development in the wasp Nasonia.

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 caudal mRNA. 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.

Localized maternal orthodenticle patterns anterior and posterior in the long germ wasp Nasonia.

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.

Somite formation: where left meets right.

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.

Genetic analysis of interactions between the somitic muscle, cartilage and tendon cell lineages during mouse development.

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.

FGF acts directly on the somitic tendon progenitors through the Ets transcription factors Pea3 and Erm to regulate scleraxis expression.

During somite development, a fibroblast growth factor (FGF) signal secreted from the myotome induces formation of a scleraxis (Scx)-expressing tendon progenitor population in the sclerotome, at the juncture between the future lineages of muscle and cartilage. While overexpression studies show that the entire sclerotome is competent to express Scx in response to FGF signaling, the normal Scx expression domain includes only the anterior and posterior dorsal sclerotome. To understand the molecular basis for this restriction, we examined the expression of a set of genes involved in FGF signaling and found that several members of the Fgf8 synexpression group are co-expressed with Scx in the dorsal sclerotome. Of particular interest were the Ets transcription factors Pea3 and Erm, which function as transcriptional effectors of FGF signaling. We show here that transcriptional activation by Pea3 and Erm in response to FGF signaling is both necessary and sufficient for Scx expression in the somite, and propose that the domain of the somitic tendon progenitors is regulated both by the restricted expression of Pea3 and Erm, and by the precise spatial relationship between these Ets transcription factors and the FGF signal originating in the myotome.

A somitic compartment of tendon progenitors.

We demonstrate that the tendons associated with the axial skeleton derive from a heretofore unappreciated, fourth compartment of the somites. Scleraxis (Scx), a bHLH transcription factor, marks this somitic tendon progenitor population at its inception, and is continuously expressed through differentiation into the mature tendons. Two earlier-formed somitic compartments, the sclerotome and myotome, interact to establish this fourth Scx-positive compartment. The tendon progenitors are induced at the sclerotome's edge, at the expense of skeletogenic Pax1 positive cells and in response to FGF signaling in the adjacent myotome. The tendon primordia thus form in a location abutting the two tissues that the mature tendons must ultimately connect. Tendon progenitor formation may reveal a general mechanism for the specification of other somitic subcompartments.

Developmental regulation of somite derivatives: muscle, cartilage and tendon.

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.