Heterozygous mutations of COL2A1 create several clinical entities collectively termed type II collagenopathies. These disorders not only impair skeletal growth but also cause ocular and otolaryngological abnormalities. The classical phenotypes include the spondyloepiphyseal dysplasia (SED) spectrum with variable severity, Stickler dysplasia type I (STD-I), and Kniest dysplasia (KND). Most COL2A1 mutations occur in the triple helical region of alpha 1(II) chains: the SED spectrum is mostly attributed to missense mutations that substitute bulky amino acids for glycine residues, STD-I to haploinsufficiency of truncation mutations, and KND to exon skipping due to splice-site mutations. To further elucidate the genotype-phenotype relationship of type II collagenopathies, we examined COL2A1 mutations in 56 families that were suspected of having type II collagenopathies, and found 38 mutations in 41 families. Phenotypes for all 22 missense mutations and one in-frame deletion in the triple helical region fell along the SED spectrum. Glycine to serine substitutions resulted in alternating zones that produce severer and milder skeletal phenotypes. Glycine to nonserine residue substitutions exclusively created more severe phenotypes. The gradient of the SED spectrum did not necessarily correlate with the occurrence of extraskeletal manifestations. All nine truncation or splice-site mutations in the triple helical or N-propeptide region caused STD-I or KND, and extraskeletal changes were inevitable in both phenotypes. All six C-propeptide mutations produced a range of atypical skeletal phenotypes and created ocular, but not otolaryngological, changes.

The Drosophila circadian clock consists of integrated autoregulatory feedback loops, making the clock difficult to elucidate without comprehensively identifying the network components in vivo. Previous studies have adopted genome-wide screening for clock-controlled genes using high-density oligonucleotide arrays that identified hundreds of clock-controlled genes. In an attempt to identify the core clock genes among these candidates, we applied genome-wide functional screening using an RNA interference (RNAi) system in vivo. Here we report the identification of novel clock gene candidates including clockwork orange (cwo), a transcriptional repressor belonging to the basic helix-loop-helix ORANGE family. cwo is rhythmically expressed and directly regulated by CLK-CYC through canonical E-box sequences. A genome-wide search for its target genes using the Drosophila genome tiling array revealed that cwo forms its own negative feedback loop and directly suppresses the expression of other clock genes through the E-box sequence. Furthermore, this negative transcriptional feedback loop contributes to sustaining a high-amplitude circadian oscillation in vivo. Based on these results, we propose that the competition between cyclic CLK-CYC activity and the adjustable threshold imposed by CWO keeps E-box-mediated transcription within the controllable range of its activity, thereby rendering a Drosophila circadian clock capable of generating high-amplitude oscillation.

The continuous usage of pyrethroids against insects has provoked the emergence of insecticide resistance that has become a major obstacle to disease vector control. The knockdown resistance (kdr) voltage-gated sodium channel gene is regarded as a key to understanding the mechanism of resistance to pyrethroids. The main purpose of this study is to identify point mutations in the sodium channel gene associated with deltamethrin resistance in Aedes aegypti. Two mutations in the IIS6 domain of the channel, S989P and V1016G, were identified as possible candidates responsible for the emergence of deltamethrin resistance in Ae. aegypti Khu Bua strain. As S989P and V1016G mutations are located within the IIS5-S6 loop and IIS6 near the ion filter and binding site, these mutations might enhance pyrethroid resistance. Allelic variation in the sodium channel gene is thought to be one of the principal molecular mechanisms regulating pyrethroid resistance in mosquitoes.

SYNOPSIS Nearly all organisms exhibit time dependent behavior and physiology across a 24 hour day known as circadian rhythms. These outputs are manifestations of endogenous cyclic gene expression patterns driven by the activity of a core transcription\translation feedback loop (TTFL). The TTFL is highly conserved across species and present in almost all mammalian cell types. This mechanism consists of a forward arm that drives gene expression and a negative arm that feeds back and inhibits the activity of the forward arm. Cyclic gene expression determines highly tissue specific functional activity regulating such processes as metabolic state, endocrine activity and neural excitability. Entrainment of these cellular clocks can be achieved through exogenous daily inputs such as light and food. Dysregulation of the TTFL has been shown to result in a wide range of disorders and diseases driving increased interest in circadian therapies.

SummaryProx1 plays pivotal roles during embryonic lymphatic development and maintenance of adult lymphatic systems by modulating the expression of various lymphatic endothelial cell (LEC) markers, such as vascular endothelial growth factor receptor 3 (VEGFR3). However, the molecular mechanisms by which Prox1 transactivates its target genes remain largely unknown. Here, we identified Ets-2 as a candidate molecule that regulates the functions of Prox1. Whereas Ets-2 has been implicated in angiogenesis, its roles during lymphangiogenesis have not yet been elucidated. We found that endogenous Ets-2 interacts with Prox1 in LECs. Using an in vivo model of chronic aseptic peritonitis, we found that Ets-2 enhanced inflammatory lymphangiogenesis, whereas a dominant-negative mutant of Ets-1 suppressed it. Ets-2 also enhanced endothelial migration towards VEGF-C through induction of expression of VEGFR3 in collaboration with Prox1. Furthermore, we found that both Prox1 and Ets-2 bind to the VEGFR3 promoter in intact chromatin. These findings suggest that Ets family members function as transcriptional cofactors that enhance Prox1-induced lymphangiogenesis.

Little is known about molecular mechanisms that control the Drosophila circadian clock beyond the transcriptional-translational feedback regulation of clock genes as an intracellular process. In this study, Early gene at 23 (E23) was identified as a novel clock gene that encodes the membrane-bound ABC transporter that is induced by the molting hormone ecdysone. E23 expresses in pacemaker neurons in fly head, and its knockdown flies lengthened circadian period with an increased expression of the clock gene vrille. E23 and vrille responded to both ecdysone and clock signals, whereas E23 protein specifically suppressed the ecdysone response and is necessary for rhythmicity. Thus, E23 forms its own feedback loop in the ecdysone response to control circadian oscillation through ecdysone-mediated vrille expression. The ecdysone signaling pathway with E23 is essential not only in developmental stage but also for the circadian behavior in adult fly.

BackgroundAnimals exhibit circadian rhythms with a period of approximately 24 h in various physiological functions, including locomotor activity. This rhythm is controlled by an endogenous oscillatory mechanism, or circadian clock, which consists of cyclically expressed clock genes and their product proteins. cryptochrome (cry) genes are thought to be involved in the clock mechanism, and their functions have been examined extensively in holometabolous insects, but in hemimetabolous insects their role is less well understood.ResultsIn the present study, the role of cry genes was investigated using RNAi technology in a hemimetabolous insect, the cricket Gryllus bimaculatus. Using a molecular cloning approach, we obtained cDNAs for two cry genes: Drosophila-type cry1 (Gb’cry1) and mammalian-type cry2 (Gb’cry2). Gb’cry2 has six splicing variants, most of which showed rhythmic mRNA expression. Gb’cry1 RNAi treatment had only a limited effect at the behavioral and molecular levels, while Gb’cry2 RNAi had a significant effect on behavioral rhythms and molecular oscillatory machinery, alone or in combination with Gb’cry1 RNAi. In Gb’cry1/Gb’cry2 double-RNAi crickets, most clock genes showed arrhythmic expression, except for timeless, which retained clear rhythmic expression. Molecular analysis revealed that some combination of Gb’cry1 and Gb’cry2 variants suppressed CLK/CYC transcriptional activity in cultured cells.ConclusionBased on these results, we propose a new model of the cricket’s circadian clock, including a molecular oscillatory loop for Gb’cry2, which can operate independent of the Gb’per/Gb’tim loop.

Sleep behaviors are observed even in nematodes and arthropods, yet little is known about how sleep-regulatory mechanisms have emerged during evolution. Here, we report a sleep-like state in the cnidarian Hydra vulgaris with a primitive nervous organization. Hydra sleep was shaped by homeostasis and necessary for cell proliferation, but it lacked free-running circadian rhythms. Instead, we detected 4-hour rhythms that might be generated by ultradian oscillators underlying Hydra sleep. Microarray analysis in sleep-deprived Hydra revealed sleep-dependent expression of 212 genes, including cGMP-dependent protein kinase 1 (PRKG1) and ornithine aminotransferase. Sleep-promoting effects of melatonin, GABA, and PRKG1 were conserved in Hydra. However, arousing dopamine unexpectedly induced Hydra sleep. Opposing effects of ornithine metabolism on sleep were also evident between Hydra and Drosophila, suggesting the evolutionary switch of their sleep-regulatory functions. Thus, sleep-relevant physiology and sleep-regulatory components may have already been acquired at molecular levels in a brain-less metazoan phylum and reprogrammed accordingly.