Dichotomous cis-regulatory motifs mediate the maturation of the neuromuscular junction by retrograde BMP signaling.

Retrograde bone morphogenetic protein (BMP) signaling at the Drosophila neuromuscular junction (NMJ) has served as a paradigm to study TGF-ß-dependent synaptic function and maturation. Yet, how retrograde BMP signaling transcriptionally regulates these functions remains unresolved. Here, we uncover a gene network, enriched for neurotransmission-related genes, that is controlled by retrograde BMP signaling in motor neurons through two Smad-binding cis-regulatory motifs, the BMP-activating (BMP-AE) and silencer (BMP-SE) elements. Unpredictably, both motifs mediate direct gene activation, with no involvement of the BMP derepression pathway regulators Schnurri and Brinker. Genome editing of candidate BMP-SE and BMP-AE within the locus of the active zone gene bruchpilot, and a novel Ly6 gene witty, demonstrated the role of these motifs in upregulating genes required for the maturation of pre- and post-synaptic NMJ compartments. Our findings uncover how Smad-dependent transcriptional mechanisms specific to motor neurons directly orchestrate a gene network required for synaptic maturation by retrograde BMP signaling.

Retrograde BMP signaling activates neuronal gene expression through widespread deployment of a conserved BMP-responsive cis-regulatory activation element.

Retrograde Bone Morphogenetic Protein (BMP) signaling in neurons is essential for the differentiation and synaptic function of many neuronal subtypes. BMP signaling regulates these processes via Smad transcription factor activity, yet the scope and nature of Smad-dependent gene regulation in neurons are mostly unknown. Here, we applied a computational approach to predict Smad-binding cis-regulatory BMP-Activating Elements (BMP-AEs) in Drosophila, followed by transgenic in vivo reporter analysis to test their neuronal subtype enhancer activity in the larval central nervous system (CNS). We identified 34 BMP-AE-containing genomic fragments that are responsive to BMP signaling in neurons, and showed that the embedded BMP-AEs are required for this activity. RNA-seq analysis identified BMP-responsive genes in the CNS and revealed that BMP-AEs selectively enrich near BMP-activated genes. These data suggest that functional BMP-AEs control nearby BMP-activated genes, which we validated experimentally. Finally, we demonstrated that the BMP-AE motif mediates a conserved Smad-responsive function in the Drosophila and vertebrate CNS. Our results provide evidence that BMP signaling controls neuronal function by directly coordinating the expression of a battery of genes through widespread deployment of a conserved Smad-responsive cis-regulatory motif.

Pentagone internalises glypicans to fine-tune multiple signalling pathways.

Tight regulation of signalling activity is crucial for proper tissue patterning and growth. Here we investigate the function of Pentagone (Pent), a secreted protein that acts in a regulatory feedback during establishment and maintenance of BMP/Dpp morphogen signalling during Drosophila wing development. We show that Pent internalises the Dpp co-receptors, the glypicans Dally and Dally-like protein (Dlp), and propose that this internalisation is important in the establishment of a long range Dpp gradient. Pent-induced endocytosis and degradation of glypicans requires dynamin- and Rab5, but not clathrin or active BMP signalling. Thus, Pent modifies the ability of cells to trap and transduce BMP by fine-tuning the levels of the BMP reception system at the plasma membrane. In addition, and in accordance with the role of glypicans in multiple signalling pathways, we establish a requirement of Pent for Wg signalling. Our data propose a novel mechanism by which morphogen signalling is regulated.

The protein kinase LKB1 negatively regulates bone morphogenetic protein receptor signaling.

The protein kinase LKB1 regulates cell metabolism and growth and is implicated in intestinal and lung cancer. Bone morphogenetic protein (BMP) signaling regulates cell differentiation during development and tissue homeostasis. We demonstrate that LKB1 physically interacts with BMP type I receptors and requires Smad7 to promote downregulation of the receptor. Accordingly, LKB1 suppresses BMP-induced osteoblast differentiation and affects BMP signaling in Drosophila wing longitudinal vein morphogenesis. LKB1 protein expression and Smad1 phosphorylation analysis in a cohort of non-small cell lung cancer patients demonstrated a negative correlation predominantly in a subset enriched in adenocarcinomas. Lung cancer patient data analysis indicated strong correlation between LKB1 loss-of-function mutations and high BMP2 expression, and these two events further correlated with expression of a gene subset functionally linked to apoptosis and migration. This new mechanism of BMP receptor regulation by LKB1 has ramifications in physiological organogenesis and disease.

Pentagone: patrolling BMP morphogen signaling.

Orchestration of spatial organization by signaling gradients--morphogen gradients--is a fundamental principle in animal development. Despite their importance in tissue patterning and growth, the exact mechanisms underlying the establishment and maintenance of morphogen gradients are poorly understood. Our recent work on BMP (bone morphogenetic protein) morphogen signaling during wing development identified a novel protein, Pentagone (Pent), as a critical regulator of morphogen activity. In the following, we discuss the properties of Pent and its role as a feed-back loop in morphogen gradient formation.

Control of Dpp morphogen signalling by a secreted feedback regulator.

In many instances during development, morphogens specify cell fates by forming concentration gradients. In the Drosophila melanogaster wing imaginal disc, Decapentaplegic (Dpp), a bone morphogenetic protein (BMP), functions as a long-range morphogen to control patterning and growth. Dpp is secreted from a stripe of cells at the anterior-posterior compartment boundary and spreads into both compartments to generate a characteristic BMP activity gradient. Ever since the identification of the morphogen activity of Dpp in the developing wing, the system has served as a paradigm to understand how long-range gradients are established and how cells respond to such gradients. Here we reveal the tight and direct connection of these two processes with the identification and characterization of pentagone (pent), a transcriptional target of BMP signalling encoding a secreted regulator of the pathway. Absence of pent in the wing disc causes a severe contraction of the BMP activity gradient resulting in patterning and growth defects. We show that Pent interacts with the glypican Dally to control Dpp distribution and provide evidence that proper establishment of the BMP morphogen gradient requires the inbuilt feedback loop embodied by Pent.

[Photoperiodic control of melatonin synthesis in fish pineal and retina]. / Contrôle photopériodique de la synthèse de mélatonine par la rétine et l'épiphyse de poisson.

Melatonin is the time-keeping molecule of vertebrates. The daily and annual variations of its rhythmic production allow synchronizing physiological functions and behaviours to the variations of the environment. In fish, melatonin is produced by the photoreceptor cells of the retina and pineal organ. It is also synthesized by other retinal cell types of the inner nuclear and ganglion cell layers. In most of the species investigated, the melatonin rhythm displays a high-at-night profile, resulting from the circadian control of the arylalkylamine N-acetyltranferase (AANAT) activity; AANAT is the penultimate enzyme in the melatonin biosynthesis pathway. Some fish species escape the high-at-night rule in the retina, and the rhythm displays a high-at-day profile, intermediate situations being sometimes observed. This review summarizes our current knowledge on the molecular and cellular mechanisms of the rhythmic control of production of an important circadian clock messenger, underlying their plasticity.

Cloning and early expression pattern of two melatonin biosynthesis enzymes in the turbot (Scophthalmus maximus).

Melatonin biosynthesis from serotonin involves the sequential activation of the arylalkylamine N-acetyltransferase (AANAT) and hydroxyindole-O-methyltransferase (HIOMT). Photoperiod synchronizes a daily rhythm in pineal and retinal melatonin secretion through controlling AANAT activity. Teleost fish possess two Aanat, one expressed in the retina (AANAT1) and the other expressed in the pineal gland (AANAT2). We report here the full-length cloning of Aanat1, Aanat2, SmHiomt and Otx5 (orthodenticle homeobox homolog 5) in the turbot (Scophthalmus maximus, Sm), a flatfish belonging to an evolutionary recent group of Teleost. The temporal expression pattern of the genes investigated is consistent with the idea that OTX5 is needed for photoreceptor specification, and that the pineal gland differentiates before the retina. SmAanat2 expression remained pineal specific during the period of time investigated, whereas SmOtx5 and SmHiomt expressions were seen in both the retina and pineal gland. Our results do not support the existence of a second SmHiomt, as is the case for SmAanat. Neither SmAanat2 nor SmHiomt mRNAs displayed cyclic accumulation in the pineal organ of embryos and larvae maintained under a light-dark cycle from fertilization onward. This is in marked contrast with the situation observed with zebrafish Aanat2, indicating that the molecular mechanisms controlling the development of the pineal melatonin system have been modified during the evolution of Teleost.

Retinal, pineal and diencephalic expression of frog arylalkylamine N-acetyltransferase-1.

The arylalkylamine N-acetyltransferase (AANAT) is a key enzyme in the rhythmic production of melatonin. Two Aanats are expressed in Teleost fish (Aanat1 in the retina and Aanat2 in the pineal organ) but only Aanat1 is found in tetrapods. This study reports the cloning of Aanat1 from R. perezi. Transcripts were mainly expressed in the retina, diencephalon, intestine and testis. In the retina and pineal organ, Aanat1 expression was in the photoreceptor cells. Expression was also seen in ependymal cells of the 3rd ventricle and discrete cells of the suprachiasmatic area. The expression of Aanat1 in both the retina and pineal organ, and the absence of Aanat2 suggests that green frog resembles more to birds and mammals than to Teleost fish, as far as Aanat is concerned. The significance of Aanat1 in extra-pineal and extra-retinal tissues remains to be elucidated; in the diencephalon, it might be associated to the so-called deep brain photoreceptor cells.

Starting the zebrafish pineal circadian clock with a single photic transition.

The issue of what starts the circadian clock ticking was addressed by studying the developmental appearance of the daily rhythm in the expression of two genes in the zebrafish pineal gland that are part of the circadian clock system. One encodes the photopigment exorhodopsin and the other the melatonin synthesizing enzyme arylalkylamine N-acetyltransferase (AANAT2). Significant daily rhythms in AANAT2 mRNA abundance were detectable for several days after fertilization in animals maintained in a normal or reversed lighting cycle providing 12 h of light and 12 h of dark. In contrast, these rhythms do not develop if animals are maintained in constant lighting or constant darkness from fertilization. In contrast to exorhodopsin, rhythmicity of AANAT2 can be initiated by a pulse of light against a background of constant darkness, by a pulse of darkness against a background of constant lighting, or by single light-to-dark or dark-to-light transitions. Accordingly, these studies indicate that circadian clock function in the zebrafish pineal gland can be initiated by minimal photic cues, and that single photic transitions can be used as an experimental tool to dissect the mechanism that starts the circadian clock in the pineal gland.