Yesser Hadj Belgacem

Sonic hedgehog signaling is decoded by calcium spike activity in the developing spinal cord

Evolutionarily conserved hedgehog proteins orchestrate the patterning of embryonic tissues, and dysfunctions in their signaling can lead to tumorigenesis. In vertebrates, Sonic hedgehog (Shh) is essential for nervous system development, but the mechanisms underlying its action remain unclear. Early electrical activity is another developmental cue important for proliferation, migration, and differentiation of neurons. Here we demonstrate the interplay between Shh signaling and Ca2+ dynamics in the developing spinal cord. Ca2+ imaging of embryonic spinal cells shows that Shh acutely increases Ca2+ spike activity through activation of the Shh coreceptor Smoothened (Smo) in neurons. Smo recruits a heterotrimeric GTP-binding protein-dependent pathway and engages both intracellular Ca2+ stores and Ca2+ influx. The dynamics of this signaling are manifested in synchronous Ca2+ spikes and inositol triphosphate transients apparent at the neuronal primary cilium. Interaction of Shh and electrical activity modulates neurotransmitter phenotype expression in spinal neurons. These results indicate that electrical activity and second-messenger signaling mediate Shh action in embryonic spinal neurons.

Regulation of feeding behaviour and locomotor activity by takeout in Drosophila.

SUMMARY The hormonal regulation of feeding behaviour is well known in vertebrates, whereas it remains poorly understood in insects. Here, we report that the takeout gene is an essential component of nutritional homeostasis in Drosophila. takeout encodes a putative juvenile hormone (JH) binding protein and has been described as a link between circadian rhythm and feeding behaviour. However, the physiological role of takeout and its putative link to JH remain unknown. In this study, we show that takeout (to1) flies failed to adapt their food intake according to food availability and that most defects could be genetically rescued. When food is abundant, to1 are hyperphagic, yielding to hypertrophy of the fat body. When food reappears after a starvation period, to1 flies do not increase their food intake as much as wild-type flies. This defect in food intake regulation is partly based on the action of Takeout on taste neurons, because the sensitivity of to1 gustatory neurons to sugars does not increase after starvation, as in wild-type neurons. This lack of regulation is also evident at the locomotor activity, which normally increases during starvation, a behaviour related to food foraging. In addition, to1 flies lack sexual dimorphism of locomotor activity, which has previously been linked to the JH circulating level. Moreover, application of the JH analog methoprene rescues the phenotype. These results suggest that takeout plays a central role as a feeding regulator and may act by modulating the circulating JH level.

Hmgcr in the Corpus Allatum Controls Sexual Dimorphism of Locomotor Activity and Body Size via the Insulin Pathway in Drosophila

The insulin signaling pathway has been implicated in several physiological and developmental processes. In mammals, it controls expression of 3-Hydroxy-3-Methylglutaryl CoA Reductase (HMGCR), a key enzyme in cholesterol biosynthesis. In insects, which can not synthesize cholesterol de novo, the HMGCR is implicated in the biosynthesis of juvenile hormone (JH). However, the link between the insulin pathway and JH has not been established. In Drosophila, mutations in the insulin receptor (InR) decrease the rate of JH synthesis. It is also known that both the insulin pathway and JH play a role in the control of sexual dimorphism in locomotor activity. In studies here, to demonstrate that the insulin pathway and HMGCR are functionally linked in Drosophila, we first show that hmgcr mutation also disrupts the sexual dimorphism. Similarly to the InR, HMGCR is expressed in the corpus allatum (ca), which is the gland where JH biosynthesis occurs. Two p[hmgcr-GAL4] lines were therefore generated where RNAi was targeted specifically against the HMGCR or the InR in the ca. We found that RNAi-HMGCR blocked HMGCR expression, while the RNAi-InR blocked both InR and HMGCR expression. Each RNAi caused disruption of sexual dimorphism and produced dwarf flies at specific rearing temperatures. These results provide evidence: (i) that HMGCR expression is controlled by the InR and (ii) that InR and HMGCR specifically in the ca, are involved in the control of body size and sexual dimorphism of locomotor activity.

Neuroendocrine control of a sexually dimorphic behavior by a few neurons of the pars intercerebralis in Drosophila

In Drosophila, locomotor activity is sexually dimorphic and the brain area controlling this dimorphism has been mapped. The neurons of the pars intercerebralis (PI) have been suggested to participate in such differences between males and females. However, the precise physical nature of the dimorphism, the identity of the PI neurons involved, and the nature of the neuronal signal coding the dimorphism remain unknown. In this study, we used a video-tracking paradigm to characterize further the pattern of locomotor activity in Drosophila. We show that the number of activity/inactivity periods (start/stop bouts) is also sexually dimorphic, and that it can be genetically feminized in males. Moreover, the transplantation of PI neurons from a female, or of feminized PI neurons from a donor male into a receiver wild-type male is sufficient to induce the feminization of locomotor behavior, confirming that this tiny cluster of ≈10 neurons is directly responsible for the sexual dimorphism in locomotor activity. Finally, feeding males with fluvastatin, a juvenile hormone (JH) inhibitor, also led to start/stop feminization, and this effect is reversible by the simultaneous application of methoprene, a JH analog, suggesting the existence of a neuroendocrine control, by JH, of such behavioral dimorphism.

Inversion of Sonic hedgehog action on its canonical pathway by electrical activity

Significance Morphogenic proteins drive the formation and patterning of tissues during embryonic development. Once tissues are formed, their cells progressively differentiate to perform the required specialized functions of the maturing tissue. Whether this transition is accompanied by changes in morphogen signaling remains unclear. Here we identify a striking inversion in Sonic hedgehog (Shh) action on its canonical Gli-dependent pathway driven by the emerging electrical activity in differentiating spinal neurons. This mechanism may allow for switching off Shh proliferative action and thus may prevent pediatric brain tumor formation and occurrence of neurodevelopmental defects. Sonic hedgehog (Shh) is a morphogenic protein that operates through the Gli transcription factor-dependent canonical pathway to orchestrate normal development of many tissues. Because aberrant levels of Gli activity lead to a wide spectrum of diseases ranging from neurodevelopmental defects to cancer, understanding the regulatory mechanisms of Shh canonical pathway is paramount. During early stages of spinal cord development, Shh specifies neural progenitors through the canonical signaling. Despite persistence of Shh as spinal cord development progresses, Gli activity is switched off by unknown mechanisms. In this study we find that Shh inverts its action on Gli during development. Strikingly, Shh decreases Gli signaling in the embryonic spinal cord by an electrical activity- and cAMP-dependent protein kinase-mediated pathway. The inhibition of Gli activity by Shh operates at multiple levels. Shh promotes cytosolic over nuclear localization of Gli2, induces Gli2 and Gli3 processing into repressor forms, and activates cAMP-responsive element binding protein that in turn represses gli1 transcription. The regulatory mechanisms identified in this study likely operate with different spatiotemporal resolution and ensure effective down-regulation of the canonical Shh signaling as spinal cord development progresses. The developmentally regulated intercalation of electrical activity in the Shh pathway may represent a paradigm for switching from canonical to noncanonical roles of developmental cues during neuronal differentiation and maturation.

Electrical activity as a developmental regulator in the formation of spinal cord circuits

Spinal cord development is a complex process involving generation of the appropriate number of cells, acquisition of distinctive phenotypes and establishment of functional connections that enable execution of critical functions such as sensation and locomotion. Here we review the basic cellular events occurring during spinal cord development, highlighting studies that demonstrate the roles of electrical activity in this process. We conclude that the participation of different forms of electrical activity is evident from the beginning of spinal cord development and intermingles with other developmental cues and programs to implement dynamic and integrated control of spinal cord function.

Spatiotemporal integration of developmental cues in neural development

Nervous system development relies on the generation of neurons, their differentiation and establishment of synaptic connections. These events exhibit remarkable plasticity and are regulated by many developmental cues. Here, we review the mechanisms of three classes of these cues: morphogenetic proteins, electrical activity, and the environment. We focus on second messenger dynamics and their role as integrators of the action of diverse cues, enabling plasticity in the process of neural development.

Crosstalk among electrical activity, trophic factors and morphogenetic proteins in the regulation of neurotransmitter phenotype specification

Morphogenetic proteins are responsible for patterning the embryonic nervous system by enabling cell proliferation that will populate all the neural structures and by specifying neural progenitors that imprint different identities in differentiating neurons. The adoption of specific neurotransmitter phenotypes is crucial for the progression of neuronal differentiation, enabling neurons to connect with each other and with target tissues. Preliminary neurotransmitter specification originates from morphogen-driven neural progenitor specification through the combinatorial expression of transcription factors according to morphogen concentration gradients, which progressively restrict the identity that born neurons adopt. However, neurotransmitter phenotype is not immutable, instead trophic factors released from target tissues and environmental stimuli change expression of neurotransmitter-synthesizing enzymes and specific vesicular transporters modifying neuronal neurotransmitter identity. Here we review studies identifying the mechanisms of catecholaminergic, GABAergic, glutamatergic, cholinergic and serotonergic early specification and of the plasticity of these neurotransmitter phenotypes during development and in the adult nervous system. The emergence of spontaneous electrical activity in developing neurons recruits morphogenetic proteins in the process of neurotransmitter phenotype plasticity, which ultimately equips the nervous system and the whole organism with adaptability for optimal performance in a changing environment.

Leukotriene B4 activates intracellular calcium and augments human osteoclastogenesis.

IntroductionBone erosion in inflammatory arthritis depends on the recruitment and activation of bone resorbing cells, the osteoclasts. Interleukin-23 (IL-23) has been primarily implicated in mediating inflammatory bone loss via the differentiation of Th17 receptor activator of nuclear factor κB ligand (RANKL)–producing cells. In this article, we describe a new role of IL-23 in activating the synthesis and production of leukotriene B4 (LTB4) in innate immune cells.MethodsWe utilized whole blood–derived human peripheral blood mononuclear cells (PBMCs), differentiated them towards an osteoclast lineage and then performed immunofluorescence and cytochemical staining to detect the expression of LTB4-associated receptors and enzymes such as phospholipase A2, 5-lipoxygenase and leukotriene A4 hydrolase, as well as the presence of tartrate-resistant acid phosphatase (TRAP) and F-actin rings on fully mature osteoclasts. We used enzyme immunoassays to measure LTB4 levels in culture media derived from IL-23-treated human PBMCs. We used real-time calcium imaging to study the effect of leukotrienes and requirements of different calcium sources and signaling proteins in activating intracellular calcium flux using pharmacological inhibitors to phospholipase C (U73122), membrane calcium channels (2-APB) and phosphatidylinositol 3-kinase (Wortmannin) and utilized qPCR for gene expression analysis in macrophages and osteoclasts.ResultsOur data show that LTB4 engagement of BLT1 and BLT2 receptors on osteoclast precursors leads to activation of phospholipase C and calcium release–activated channel–mediated intracellular calcium flux, which can activate further LTB4 autocrine production. IL-23-induced synthesis and secretion of LTB4 resulted in the upregulation of osteoclast-related genes NFATC1, MMP9, ACP5, CTSK and ITGB3 and the formation of giant, multinucleated TRAP+ cells capable of F-actin ring formation. These effects were dependent on Ca2+ signaling and were completely inhibited by BLT1/BLT2 and/or PLC and CRAC inhibitors.ConclusionsIn conclusion, IL-23 can initiate osteoclast differentiation independently from the RANK-RANKL pathway by utilizing Ca2+ signaling and the LTB4 signaling cascade.

The Many Hats of Sonic Hedgehog Signaling in Nervous System Development and Disease

Sonic hedgehog (Shh) signaling occurs concurrently with the many processes that constitute nervous system development. Although Shh is mostly known for its proliferative and morphogenic action through its effects on neural stem cells and progenitors, it also contributes to neuronal differentiation, axonal pathfinding and synapse formation and function. To participate in these diverse events, Shh signaling manifests differently depending on the maturational state of the responsive cell, on the other signaling pathways regulating neural cell function and the environmental cues that surround target cells. Shh signaling is particularly dynamic in the nervous system, ranging from canonical transcription-dependent, to non-canonical and localized to axonal growth cones. Here, we review the variety of Shh functions in the developing nervous system and their consequences for neurodevelopmental diseases and neural regeneration, with particular emphasis on the signaling mechanisms underlying Shh action.