Osteocytes and Weightlessness.

PURPOSE OF REVIEW: Osteocytes are considered to be the cells responsible for mastering the remodeling process that follows the exposure to unloading conditions. Given the invasiveness of bone biopsies in humans, both rodents and in vitro culture systems are largely adopted as models for studies in space missions or in simulated microgravity conditions models on Earth. RECENT FINDINGS: After a brief recall of the main changes in bone mass and osteoclastic and osteoblastic activities in space-related models, this review focuses on the potential role of osteocytes in directing these changes. The role of the best-known signalling molecules is questioned, in particular in relation to osteocyte apoptosis. The mechanotransduction actors identified in spatial conditions and the problems related to fluid flow and shear stress changes, probably enhanced by the alteration in fluid flow and lack of convection during spaceflight, are recalled and discussed.

Gut Microbiota and Host Juvenile Growth.

Good genes, good food, good friends. That is what parents hope will sustain and nurture the harmonious growth of their children. The impact of the genetic background and nutrition on postnatal growth has been in the spot light for long, but the good friends have come to the scene only recently. Among the good friends perhaps the most crucial ones are those that we are carrying within ourselves. They comprise the trillions of microbes that collectively constitute each individual's intestinal microbiota. Indeed, recent epidemiological and field studies in humans, supported by extensive experimental data on animal models, demonstrate a clear role of the intestinal microbiota on their host's juvenile growth, especially under suboptimal nutrient conditions. Genuinely integrative approaches applicable to invertebrate and vertebrate systems combine tools from genetics, developmental biology, microbiology, nutrition, and physiology to reveal how gut microbiota affects growth both positively and negatively, in healthy and pathological conditions. It appears that certain natural or engineered gut microbiota communities can positively impact insulin/IGF-1 and steroid hormone signaling, thus contributing to the host juvenile development and maturation.

Btk-dependent epithelial cell rearrangements contribute to the invagination of nearby tubular structures in the posterior spiracles of Drosophila.

The Drosophila respiratory system consists of two connected organs, the tracheae and the spiracles. Together they ensure the efficient delivery of air-borne oxygen to all tissues. The posterior spiracles consist internally of the spiracular chamber, an invaginated tube with filtering properties that connects the main tracheal branch to the environment, and externally of the stigmatophore, an extensible epidermal structure that covers the spiracular chamber. The primordia of both components are first specified in the plane of the epidermis and subsequently the spiracular chamber is internalized through the process of invagination accompanied by apical cell constriction. It has become clear that invagination processes do not always or only rely on apical constriction. We show here that in mutants for the src-like kinase Btk29A spiracle cells constrict apically but do not complete invagination, giving rise to shorter spiracular chambers. This defect can be rescued by using different GAL4 drivers to express Btk29A throughout the ectoderm, in cells of posterior segments only, or in the stigmatophore pointing to a non cell-autonomous role for Btk29A. Our analysis suggests that complete invagination of the spiracular chamber requires Btk29A-dependent planar cell rearrangements of adjacent non-invaginating cells of the stigmatophore. These results highlight the complex physical interactions that take place among organ components during morphogenesis, which contribute to their final form and function.

A surfactant lipid layer of endosomal membranes facilitates airway gas filling in Drosophila.

The mechanisms underlying the construction of an air-liquid interface in respiratory organs remain elusive. Here, we use live imaging and genetic analysis to describe the morphogenetic events generating an extracellular lipid lining of the Drosophila airways required for their gas filing and animal survival. We show that sequential Rab39/Syx1A/Syt1-mediated secretion of lysosomal acid sphingomyelinase (Drosophila ASM [dASM]) and Rab11/35/Syx1A/Rop-dependent exosomal secretion provides distinct components for lipid film assembly. Tracheal inactivation of Rab11 or Rab35 or loss of Rop results in intracellular accumulation of exosomal, multi-vesicular body (MVB)-derived vesicles. On the other hand, loss of dASM or Rab39 causes luminal bubble-like accumulations of exosomal membranes and liquid retention in the airways. Inactivation of the exosomal secretion in dASM mutants counteracts this phenotype, arguing that the exosomal secretion provides the lipid vesicles and that secreted lysosomal dASM organizes them into a continuous film. Our results reveal the coordinated functions of extracellular vesicle and lysosomal secretions in generating a lipid layer crucial for airway gas filling and survival.

Treatment with fibroblast growth factor 19 increases skeletal muscle fiber size, ameliorates metabolic perturbations and hepatic inflammation in 5/6 nephrectomized mice.

Chronic kidney disease (CKD) is associated with osteosarcopenia, and because a physical decline in patients correlates with an increased risk of morbidity, an improvement of the musculoskeletal system is expected to improve morbi-mortality. We recently uncovered that the intestinal hormone Fibroblast Growth Factor 19 (FGF19) is able to promote skeletal muscle mass and strength in rodent models, in addition to its capacity to improve glucose homeostasis. Here, we tested the effects of a treatment with recombinant human FGF19 in a CKD mouse model, which associates sarcopenia and metabolic disorders. In 5/6 nephrectomized (5/6Nx) mice, subcutaneous FGF19 injection (0.1 mg/kg) during 18 days increased skeletal muscle fiber size independently of food intake and weight gain, associated with decreased gene expression of myostatin. Furthermore, FGF19 treatment attenuated glucose intolerance and reduced hepatic expression of gluconeogenic genes in uremic mice. Importantly, the treatment also decreased gene expression of liver inflammatory markers in CKD mice. Therefore, our results suggest that FGF19 may represent a novel interesting therapeutic strategy for a global improvement of sarcopenia and metabolic complications in CKD.

Positional cues in the Drosophila nerve cord: semaphorins pattern the dorso-ventral axis.

During the development of neural circuitry, neurons of different kinds establish specific synaptic connections by selecting appropriate targets from large numbers of alternatives. The range of alternative targets is reduced by well organised patterns of growth, termination, and branching that deliver the terminals of appropriate pre- and postsynaptic partners to restricted volumes of the developing nervous system. We use the axons of embryonic Drosophila sensory neurons as a model system in which to study the way in which growing neurons are guided to terminate in specific volumes of the developing nervous system. The mediolateral positions of sensory arbors are controlled by the response of Robo receptors to a Slit gradient. Here we make a genetic analysis of factors regulating position in the dorso-ventral axis. We find that dorso-ventral layers of neuropile contain different levels and combinations of Semaphorins. We demonstrate the existence of a central to dorsal and central to ventral gradient of Sema 2a, perpendicular to the Slit gradient. We show that a combination of Plexin A (Plex A) and Plexin B (Plex B) receptors specifies the ventral projection of sensory neurons by responding to high concentrations of Semaphorin 1a (Sema 1a) and Semaphorin 2a (Sema 2a). Together our findings support the idea that axons are delivered to particular regions of the neuropile by their responses to systems of positional cues in each dimension.

Ketogenic diet administration to mice after a high-fat-diet regimen promotes weight loss, glycemic normalization and induces adaptations of ketogenic pathways in liver and kidney.

OBJECTIVE: The ketogenic diet (KD), characterized by very limited dietary carbohydrate intake and used as nutritional treatment for GLUT1-deficiency syndromes and pharmacologically refractory epilepsy, may promote weight loss and improve metabolic fitness, potentially alleviating the symptoms of osteoarthritis. Here, we have studied the effects of administration of a ketogenic diet in mice previously rendered obese by feeding a high fat diet (HFD) and submitted to surgical destabilization of the medial meniscus to mimic osteoarthritis. METHODS: 6-weeks old mice were fed an HFD for 10 weeks and then switched to a chow diet (CD), KD or maintained on a HFD for 8 weeks. Glycemia, ß-hydroxybutyrate (BHB), body weight and fat mass were compared among groups. In liver and kidney, protein expression and histone post-translational modifications were assessed by Western blot, and gene expression by quantitative Real-Time PCR. RESULTS: After a 10 weeks HDF feeding, administration for 8 weeks of a KD or CD induced a comparable weight loss and decrease in fat mass, with better glycemic normalization in the KD group. Histone ß-hydroxybutyrylation, but not histone acetylation, was increased in the liver and kidney of mice fed the KD and the rate-limiting ketogenic enzyme HMGCS2 was upregulated - at the gene and protein level - in liver and, to an even greater extent, in kidney. KD-induced HMGCS2 overexpression may be dependent on FGF21, whose gene expression was increased by KD in liver. CONCLUSIONS: Over a period of 8 weeks, KD is more effective than a chow diet to induce metabolic normalization. Besides acting as a fuel molecule, BHB may exert its metabolic effects through modulation of the epigenome - via histone ß-hydroxybutyrylation - and extensive transcriptional modulation in liver and kidney.

The Skeletal Cellular and Molecular Underpinning of the Murine Hindlimb Unloading Model.

Bone adaptation to spaceflight results in bone loss at weight bearing sites following the absence of the stimulus represented by ground force. The rodent hindlimb unloading model was designed to mimic the loss of mechanical loading experienced by astronauts in spaceflight to better understand the mechanisms causing this disuse-induced bone loss. The model has also been largely adopted to study disuse osteopenia and therefore to test drugs for its treatment. Loss of trabecular and cortical bone is observed in long bones of hindlimbs in tail-suspended rodents. Over the years, osteocytes have been shown to play a key role in sensing mechanical stress/stimulus via the ECM-integrin-cytoskeletal axis and to respond to it by regulating different cytokines such as SOST and RANKL. Colder experimental environments (~20-22°C) below thermoneutral temperatures (~28-32°C) exacerbate bone loss. Hence, it is important to consider the role of environmental temperatures on the experimental outcomes. We provide insights into the cellular and molecular pathways that have been shown to play a role in the hindlimb unloading and recommendations to minimize the effects of conditions that we refer to as confounding factors.

Drosophila Perpetuates Nutritional Mutualism by Promoting the Fitness of Its Intestinal Symbiont Lactobacillus plantarum.

Facultative animal-bacteria symbioses, which are critical determinants of animal fitness, are largely assumed to be mutualistic. However, whether commensal bacteria benefit from the association has not been rigorously assessed. Using a simple and tractable gnotobiotic model- Drosophila mono-associated with one of its dominant commensals, Lactobacillus plantarum-we reveal that in addition to benefiting animal growth, this facultative symbiosis has a positive impact on commensal bacteria fitness. We find that bacteria encounter a strong cost during gut transit, yet larvae-derived maintenance factors override this cost and increase bacterial population fitness, thus perpetuating symbiosis. In addition, we demonstrate that the maintenance of the association is required for achieving maximum animal growth benefits upon chronic undernutrition. Taken together, our study establishes a prototypical case of facultative nutritional mutualism, whereby a farming mechanism perpetuates animal-bacteria symbiosis, which bolsters fitness gains for both partners upon poor nutritional conditions.

The role of the microbial environment in Drosophila post-embryonic development.

Development, growth and maturation of animals are under genetic and environmental control. Multicellular organisms interact throughout their lives with a variety of environment- and body-associated microorganisms. It has now been appreciated that the very conspicuous and varied microbial population associated with the food and the gastro-intestinal tract is a critical factor that can influence growth. Beyond the phenomenology, the mechanisms underlying the beneficial effects of microbes on development are being revealed from studies in Drosophila melanogaster, a particularly well suited system for a mechanistic understanding of host/microbiota interactions. Association of otherwise germ-free eggs with specific bacterial strains isolated from Drosophila gut samples can accelerate growth in larvae raised on restrictive diets. We review advances made possible by the exploitation of such simplified gnotobiotic systems in the search for the genes, molecules and physiological adaptations responsible for this effect in both host and microbes. Transposon mutagenesis and gene-trait match studies in bacteria can identify the key microbial genes and metabolites required for the beneficial effect, acetic acid being one of them. In the fly, functional genomic analysis, transcriptomics and metabolomics point to the modulation of systemic insulin and steroid hormone signalling as well as the regulation of intestinal physiology, including the enhancement of intestinal protease activity, as crucial mediators of the host's response.

The immunoglobulin superfamily of neuronal cell adhesion molecules: lessons from animal models and correlation with human disease.

Neuronal cell adhesion molecules of the immunoglobulin superfamily (IgCAMs) play a crucial role in the formation of neural circuits at different levels: cell migration, axonal and dendritic targeting as well as synapse formation. Furthermore, in perinatal and adult life, neuronal IgCAMs are required for the formation and maintenance of specialized axonal membrane domains, synaptic plasticity and neurogenesis. Mutations in the corresponding human genes have been correlated to several human neuronal disorders. Perturbing neuronal IgCAMs in animal models provides powerful means to understand the molecular and cellular basis of such human disorders. In this review, we concentrate on the NCAM, L1 and contactin subfamilies of neuronal IgCAMs summarizing recent functional studies from model systems and highlighting their links to disease pathogenesis.

The IgLON protein Lachesin is required for the blood-brain barrier in Drosophila.

In the mammalian peripheral nervous system, nerve insulation depends on the integrity of paranodal junctions between axons and their ensheathing glia. Ultrastructurally, these junctions are similar to the septate junctions (SJ) of invertebrates. In Drosophila, SJ are found in epithelia and in the glia that form the blood-brain barrier (BBB). Drosophila NeurexinIV and Gliotactin, two components of SJ, play an important role in nerve ensheathment and insulation. Here, we report that Drosophila Lachesin (Lac), another SJ component, is also required for a functional BBB. In the developing nervous system, Lac is expressed in a dynamic pattern by surface glia and a subset of neurons. Ultrastructural analysis of Lac mutant embryos shows poorly developed SJ in surface glia and epithelia where Lac is expressed. Mutant embryos undergo a phase of hyperactivity, with unpatterned muscle contractions, and subsequently become paralyzed and fail to hatch. We propose that this phenotype reflects a failure in BBB function.

Mechanisms of morphogen movement.

Morphogens are defined as signaling molecules that are produced locally, yet act directly at a distance to pattern the surrounding field of cells in a concentration-dependent manner. In recent years many laboratories have devoted their attention to how morphogens actually reach distant cells. Several models have been proposed, including diffusion in the extracellular space and planar transcytosis. A combination of genetic, developmental, and cell-biological approaches have been taken to tackle this issue. I will present the models and discuss the types of experiments that have been designed to test them. It stands out that most of the work has been carried out in Drosophila. Morphogens contribute to patterning of the vertebrate nervous system, and the same signaling molecules have recently been shown to play important, possibly instructive, roles in axon guidance. Little, if anything, is known about the movement of morphogens in the context of nervous system development. The long-standing tradition of biophysical studies on diffusion in the brain extracellular space, along with the sophisticated in vitro culture systems developed in neurobiology laboratories, may provide new tools and ideas to test these models in a new context.

Lachesin is a component of a septate junction-based mechanism that controls tube size and epithelial integrity in the Drosophila tracheal system.

Organ morphogenesis requires the coordinated activity of many mechanisms involved in cell rearrangements, size control, cell proliferation and organ integrity. Here we report that Lachesin (Lac), a cell surface protein, is required for the proper morphogenesis of the Drosophila tracheal system. Homozygous embryos for Lac mutations, which we find fail to complement the previous identified bulbous (bulb) mutation, display convoluted tracheal tubes and tube breaks. At the cellular level, we can detect enlarged cells, suggesting that Lac regulates organ size by influencing cell length rather than cell number, and cell detachments, indicating a role for Lac in cell adhesion. Results from an in vitro assay further support that Lac behaves as a homophilic cell adhesion molecule. Lac co-localizes with Septate Junction (SJ) proteins, and ultrastructural analysis confirms that it accumulates specifically at this type of cellular junction. In Lac mutant embryos, previously characterized components of the SJs are mislocalized, indicating that the proper organization of SJs requires Lac function. In addition, mutations in genes encoding other components of the SJs produce a similar tracheal phenotype. These results point out a new role of the SJs in morphogenesis regulating cell adhesion and cell size.