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Plant trichomes originate from epidermal cell, forming protective structure from abiotic and biotic stresses. Different from the unicellular trichome in Arabidopsis, tomato trichomes are multicellular structure and can be classified into seven different types based on cell number, shape and the presence of glandular cells. Despite the importance of tomato trichomes in insect resistance, our understanding of the tomato trichome morphogenesis remains elusive. In this study, we quantitatively analyzed morphological traits of trichomes in tomato and further performed live imaging of cytoskeletons in stably transformed lines with actin and microtubule markers. At different developmental stages, two types of cytoskeletons exhibited distinct patterns in different trichome cells, ranging from transverse, spiral to longitudinal. This gradual transition of actin filament angle from basal to top cells could correlate with the spatial expansion mode in different cells. Further genetic screen for aberrant trichome morphology led to the discovery of a number of independent mutations in SCAR/WAVE and ARP2/3 complex, which resulted in actin bundling and distorted trichomes. Disruption of microtubules caused isotropic expansion while abolished actin filaments entirely inhibited axial extension of trichomes, indicating that microtubules and actin filaments may control distinct aspects of trichome cell expansion. Our results shed light on the roles of cytoskeletons in the formation of multicellular structure of tomato trichomes. [ABSTRACT FROM AUTHOR]

Neuronal physiology is particularly sensitive to acute stressors that affect excitability, many of which can trigger seizures and epilepsies. Although intrinsic neuronal homeostasis plays an important role in maintaining overall nervous system robustness and its resistance to stressors, the specific genetic and molecular mechanisms that underlie these processes are not well understood. Here we used a reverse genetic approach in Drosophila to test the hypothesis that specific voltage-gated ion channels contribute to neuronal homeostasis, robustness, and stress resistance. We found that the activity of the voltage-gated potassium channel seizure (sei), an ortholog of the mammalian ERG channel family, is essential for protecting flies from acute heat-induced seizures. Although sei is broadly expressed in the nervous system, our data indicate that its impact on the organismal robustness to acute environmental stress is primarily mediated via its action in excitatory neurons, the octopaminergic system, as well as neuropile ensheathing and perineurial glia. Furthermore, our studies suggest that human mutations in the human ERG channel (hERG), which have been primarily implicated in the cardiac Long QT Syndrome (LQTS), may also contribute to the high incidence of seizures in LQTS patients via a cardiovascular-independent neurogenic pathway. [ABSTRACT FROM AUTHOR]

The control of the size of internal tubular organs, such as the lungs or vascular system, is critical for proper physiological activity and to prevent disease or malformations. This control incorporates the intrinsic physical anisotropy of tubes to generate proportionate organs that match their function. The exact mechanisms underlying tube size control and how tubular anisotropy is translated at the cellular level are still not fully understood. Here we investigate these mechanisms using the Drosophila tracheal system. We show that the apical polarity protein Crumbs transiently accumulates anisotropically at longitudinal cell junctions during tube elongation. We provide evidence indicating that the accumulation of Crumbs in specific apical domains correlates with apical surface expansion, suggesting a link between the anisotropic accumulation of Crumbs at the cellular level and membrane expansion. We find that Src42A is required for the anisotropic accumulation of Crumbs, thereby identifying the first polarised cell behaviour downstream of Src42A. Our results indicate that Src42A regulates a mechanism that increases the fraction of Crb protein at longitudinal junctions, and genetic interaction experiments are consistent with Crb acting downstream of Src42A in controlling tube size. Collectively, our results suggest a model in which Src42A would sense the inherent anisotropic mechanical tension of the tube and translate it into a polarised Crumbs accumulation, which may promote a bias towards longitudinal membrane expansion, orienting cell elongation and, as a consequence, longitudinal growth at the tissue level. This work provides new insights into the key question of how organ growth is controlled and polarised and unveils the function of two conserved proteins, Crumbs and Src42A, with important roles in development and homeostasis as well as in disease, in this biological process. [ABSTRACT FROM AUTHOR]

The circadian clock coordinates physiology and metabolism. mTOR (mammalian/mechanistic target of rapamycin) is a major intracellular sensor that integrates nutrient and energy status to regulate protein synthesis, metabolism, and cell growth. Previous studies have identified a key role for mTOR in regulating photic entrainment and synchrony of the central circadian clock in the suprachiasmatic nucleus (SCN). Given that mTOR activities exhibit robust circadian oscillations in a variety of tissues and cells including the SCN, here we continued to investigate the role of mTOR in orchestrating autonomous clock functions in central and peripheral circadian oscillators. Using a combination of genetic and pharmacological approaches we show that mTOR regulates intrinsic clock properties including period and amplitude. In peripheral clock models of hepatocytes and adipocytes, mTOR inhibition lengthens period and dampens amplitude, whereas mTOR activation shortens period and augments amplitude. Constitutive activation of mTOR in Tsc2–/–fibroblasts elevates levels of core clock proteins, including CRY1, BMAL1 and CLOCK. Serum stimulation induces CRY1 upregulation in fibroblasts in an mTOR-dependent but Bmal1- and Period-independent manner. Consistent with results from cellular clock models, mTOR perturbation also regulates period and amplitude in the ex vivo SCN and liver clocks. Further, mTOR heterozygous mice show lengthened circadian period of locomotor activity in both constant darkness and constant light. Together, these results support a significant role for mTOR in circadian timekeeping and in linking metabolic states to circadian clock functions. [ABSTRACT FROM AUTHOR]

The growth plate mediates bone growth where SOX9 and GLI factors control chondrocyte proliferation, differentiation and entry into hypertrophy. FOXA factors regulate hypertrophic chondrocyte maturation. How these factors integrate into a Gene Regulatory Network (GRN) controlling these differentiation transitions is incompletely understood. We adopted a genome-wide whole tissue approach to establish a rowth late ifferential ene xpression ibrary (GP-DGEL) for fractionated proliferating, pre-hypertrophic, early and late hypertrophic chondrocytes, as an overarching resource for discovery of pathways and disease candidates. De novo motif discovery revealed the enrichment of SOX9 and GLI binding sites in the genes preferentially expressed in proliferating and prehypertrophic chondrocytes, suggesting the potential cooperation between SOX9 and GLI proteins. We integrated the analyses of the transcriptome, SOX9, GLI1 and GLI3 ChIP-seq datasets, with functional validation by transactivation assays and mouse mutants. We identified new SOX9 targets and showed SOX9-GLI directly and cooperatively regulate many genes such as Trps1, Sox9, Sox5, Sox6, Col2a1, Ptch1, Gli1 and Gli2. Further, FOXA2 competes with SOX9 for the transactivation of target genes. The data support a model of SOX9-GLI-FOXA phasic GRN in chondrocyte development. Together, SOX9-GLI auto-regulate and cooperate to activate and repress genes in proliferating chondrocytes. Upon hypertrophy, FOXA competes with SOX9, and control toward terminal differentiation passes to FOXA, RUNX, AP1 and MEF2 factors. [ABSTRACT FROM AUTHOR]

We here report on the existence of Leber’s hereditary optic neuropathy (LHON) associated with peculiar combinations of individually non-pathogenic missense mitochondrial DNA (mtDNA) variants, affecting the MT-ND4, MT-ND4L and MT-ND6 subunit genes of Complex I. The pathogenic potential of these mtDNA haplotypes is supported by multiple evidences: first, the LHON phenotype is strictly inherited along the maternal line in one very large family; second, the combinations of mtDNA variants are unique to the two maternal lineages that are characterized by recurrence of LHON; third, the Complex I-dependent respiratory and oxidative phosphorylation defect is co-transferred from the proband’s fibroblasts into the cybrid cell model. Finally, all but one of these missense mtDNA variants cluster along the same predicted fourth E-channel deputed to proton translocation within the transmembrane domain of Complex I, involving the ND1, ND4L and ND6 subunits. Hence, the definition of the pathogenic role of a specific mtDNA mutation becomes blurrier than ever and only an accurate evaluation of mitogenome sequence variation data from the general population, combined with functional analyses using the cybrid cell model, may lead to final validation. Our study conclusively shows that even in the absence of a clearly established LHON primary mutation, unprecedented combinations of missense mtDNA variants, individually known as polymorphisms, may lead to reduced OXPHOS efficiency sufficient to trigger LHON. In this context, we introduce a new diagnostic perspective that implies the complete sequence analysis of mitogenomes in LHON as mandatory gold standard diagnostic approach. [ABSTRACT FROM AUTHOR]

Integrated development of diverse tissues gives rise to a functional, mobile vertebrate musculoskeletal system. However, the genetics and cellular interactions that drive the integration of muscle, tendon, and skeleton are poorly understood. In the vertebrate head, neural crest cells, from which cranial tendons derive, pattern developing muscles just as tendons have been shown to in limb and trunk tissue, yet the mechanisms of this patterning are unknown. From a forward genetic screen, we determined that cyp26b1 is critical for musculoskeletal integration in the ventral pharyngeal arches, particularly in the mandibulohyoid junction where first and second arch muscles interconnect. Using time-lapse confocal analyses, we detail musculoskeletal integration in wild-type and cyp26b1 mutant zebrafish. In wild-type fish, tenoblasts are present in apposition to elongating muscles and condense in discrete muscle attachment sites. In the absence of cyp26b1, tenoblasts are generated in normal numbers but fail to condense into nascent tendons within the ventral arches and, subsequently, muscles project into ectopic locales. These ectopic muscle fibers eventually associate with ectopic tendon marker expression. Genetic mosaic analysis demonstrates that neural crest cells require Cyp26b1 function for proper musculoskeletal development. Using an inhibitor, we find that Cyp26 function is required in a short time window that overlaps the dynamic window of tenoblast condensation. However, cyp26b1 expression is largely restricted to regions between tenoblast condensations during this time. Our results suggest that degradation of RA by this previously undescribed population of neural crest cells is critical to promote condensation of adjacent scxa-expressing tenoblasts and that these condensations are subsequently required for proper musculoskeletal integration. [ABSTRACT FROM AUTHOR]

Small RNAs called PIWI -interacting RNAs (piRNAs) are essential for transposon control and fertility in animals. Primary processing is the small RNA biogenesis pathway that uses long single-stranded RNA precursors to generate millions of individual piRNAs, but the molecular mechanisms that identify a transcript as a precursor are poorly understood. Here we demonstrate that artificial tethering of the piRNA biogenesis factor, Armi, to a transcript is sufficient to direct it into primary processing in Drosophila ovaries and in an ovarian cell culture model. In the fly ovarian somatic follicle cells, the transcript becomes cleaved in a stepwise manner, with a 5′→3′ directionality, liberating U1-containing ~24 nt piRNAs that are loaded into Piwi. Although uridines are preferred for generation of piRNA 5′ ends, processing takes place even in their absence, albeit at a lower efficiency. We show that recombinant Armi has 5′→3′ helicase activity, and mutations that abolish this activity also reduce piRNA processing in vivo. Another somatic piRNA pathway factor Yb, an interactor of Armi, is also able to trigger piRNA biogenesis when tethered to a transcript. Tethering-mediated primary piRNA biogenesis is also functional in the fly ovarian germline and loads all the three PIWI proteins present in this environment. Our study finds a broad correlation between piRNA processing and localization of the tethered factors to the cytoplasmic perinuclear ribonucleoprotein granules called germline nuage or somatic Yb bodies. We conclude that transcripts bound by Armi and Yb are identified as piRNA precursors, resulting in localization to cytoplasmic processing granules and their subsequent engagement by the resident piRNA biogenesis machinery. [ABSTRACT FROM AUTHOR]

Understanding the cellular-molecular substrates of heart disease is key to the development of cardiac specific therapies and to the prevention of off-target effects by non-cardiac targeted drugs. One of the primary targets for therapeutic intervention has been the human ether a go-go (hERG) K+ channel that, together with the KCNQ channel, controls the rate and efficiency of repolarization in human myocardial cells. Neither of these channels plays a major role in adult mouse heart function; however, we show here that the hERG homolog seizure (sei), along with KCNQ, both contribute significantly to adult heart function as they do in humans. In Drosophila, mutations in or cardiac knockdown of sei channels cause arrhythmias that become progressively more severe with age. Intracellular recordings of semi-intact heart preparations revealed that these perturbations also cause electrical remodeling that is reminiscent of the early afterdepolarizations seen in human myocardial cells defective in these channels. In contrast to KCNQ, however, mutations in sei also cause extensive structural remodeling of the myofibrillar organization, which suggests that hERG channel function has a novel link to sarcomeric and myofibrillar integrity. We conclude that deficiency of ion channels with similar electrical functions in cardiomyocytes can lead to different types or extents of electrical and/or structural remodeling impacting cardiac output. [ABSTRACT FROM AUTHOR]

The intricate shaping of the facial skeleton is essential for function of the vertebrate jaw and middle ear. While much has been learned about the signaling pathways and transcription factors that control facial patterning, the downstream cellular mechanisms dictating skeletal shapes have remained unclear. Here we present genetic evidence in zebrafish that three major signaling pathways − Jagged-Notch, Endothelin1 (Edn1), and Bmp − regulate the pattern of facial cartilage and bone formation by controlling the timing of cartilage differentiation along the dorsoventral axis of the pharyngeal arches. A genomic analysis of purified facial skeletal precursors in mutant and overexpression embryos revealed a core set of differentiation genes that were commonly repressed by Jagged-Notch and induced by Edn1. Further analysis of the pre-cartilage condensation gene barx1, as well as in vivo imaging of cartilage differentiation, revealed that cartilage forms first in regions of high Edn1 and low Jagged-Notch activity. Consistent with a role of Jagged-Notch signaling in restricting cartilage differentiation, loss of Notch pathway components resulted in expanded barx1 expression in the dorsal arches, with mutation of barx1 rescuing some aspects of dorsal skeletal patterning in jag1b mutants. We also identified prrx1a and prrx1b as negative Edn1 and positive Bmp targets that function in parallel to Jagged-Notch signaling to restrict the formation of dorsal barx1+ pre-cartilage condensations. Simultaneous loss of jag1b and prrx1a/b better rescued lower facial defects of edn1 mutants than loss of either pathway alone, showing that combined overactivation of Jagged-Notch and Bmp/Prrx1 pathways contribute to the absence of cartilage differentiation in the edn1 mutant lower face. These findings support a model in which Notch-mediated restriction of cartilage differentiation, particularly in the second pharyngeal arch, helps to establish a distinct skeletal pattern in the upper face. [ABSTRACT FROM AUTHOR]

Heterodimers of CLOCK and BMAL1 are the major transcriptional activators of the mammalian circadian clock. Because the paralog NPAS2 can substitute for CLOCK in the suprachiasmatic nucleus (SCN), the master circadian pacemaker, CLOCK-deficient mice maintain circadian rhythms in behavior and in tissues in vivo. However, when isolated from the SCN, CLOCK-deficient peripheral tissues are reportedly arrhythmic, suggesting a fundamental difference in circadian clock function between SCN and peripheral tissues. Surprisingly, however, using luminometry and single-cell bioluminescence imaging of PER2 expression, we now find that CLOCK-deficient dispersed SCN neurons and peripheral cells exhibit similarly stable, autonomous circadian rhythms in vitro. In CLOCK-deficient fibroblasts, knockdown of Npas2 leads to arrhythmicity, suggesting that NPAS2 can compensate for loss of CLOCK in peripheral cells as well as in SCN. Our data overturn the notion of an SCN-specific role for NPAS2 in the molecular circadian clock, and instead indicate that, at the cellular level, the core loops of SCN neuron and peripheral cell circadian clocks are fundamentally similar. [ABSTRACT FROM AUTHOR]