Two eARCHT3.0 Lines for Optogenetic Silencing of Dopaminergic and Serotonergic Neurons

Dopaminergic and serotonergic neurons modulate and control processes ranging from reward signaling to regulation of motor outputs. Further, dysfunction of these neurons is involved in both degenerative and psychiatric disorders. Elucidating the roles of these neurons has been greatly facilitated by bacterial artificial chromosome (BAC) transgenic mouse lines expressing channelrhodopsin to readily enable cell-type specific activation. However, corresponding lines to silence these monoaminergic neurons have been lacking. We have generated two BAC transgenic mouse lines expressing the outward proton pump, enhanced ArchT3.0 (eArchT3.0), and GFP under control of the regulatory elements of either the dopamine transporter (DAT; Jax# 031663) or the tryptophan hydroxylase 2 (TPH2; Jax# 031662) gene locus. We demonstrate highly faithful and specific expression of these lines in dopaminergic and serotonergic neurons respectively. Additionally we validate effective and sensitive eArchT3.0-mediated silencing of these neurons using slice electrophysiology as well as with a well-established behavioral assay. These new transgenic tools will help expedite the study of dopaminergic and serotonergic system function in normal behavior and disease

Regulation of Neuronal Dendrite Development and Migration by the Atypical Cadherin Fat3

Neuronal shape and position are critical to the formation and function of neuronal circuits. Although neurons develop axons and dendrites cell-autonomously in vitro, how extracellular cues in vivo direct neurite specification and placement remains poorly understood. The role of atypical cadherin Fat3 in amacrine cell development illustrates how the same extracellular cue can guide both dendrite formation and migration. In the mouse retina, amacrine cells have a bipolar morphology during their migration. Upon reaching the nascent inner plexiform layer (IPL), they elaborate one neurite into the IPL and retract the other. Loss of Fat3 leads amacrine cells to develop an extra dendrite outside the IPL as well as errors in migration. We found Fat3 protein is concentrated at the IPL throughout amacrine cell development, suggesting Fat3 detects a directional signal. Here we investigated the signaling pathways upstream and downstream of Fat3 that mediate its role in amacrine cell development. In Drosophila, Fat’s ligand is Dachsous. Fat and Dachsous binding is modulated by the kinase Four-jointed. Our analysis of mutant retinas determined that Fat3 and vertebrate Four-jointed genetically interacted. However knockout studies of vertebrate Dachsous homologues suggested they are not relevant Fat3 ligands in the retina. Instead, analysis of retinas missing retinal ganglion cells suggested Fat3-mediated homophilic adhesion between amacrine cells may be important. Sparse loss of Fat3 from amacrine cells also led to extraneous neurites, suggesting Fat3 acts cell autonomously. Ex vivo live imaging revealed both migration and neurite dynamics were less directed in Fat3 mutant amacrine cells. We hypothesized Fat3 acts to target asymmetric localization of cytoskeletal regulators to the leading neurite. To identify downstream Fat3 effectors, we performed a pulldown assay using the Fat3 intracellular domain. We identified several cytoskeletal regulators as candidate binding partners. We focused on the actin regulators Ena/VASP and demonstrated a direct interaction with the Fat3 intracellular domain. In the retina, Ena/VASP localized with Fat3 to the IPL, and loss of Fat3 changed Ena/VASP distribution. Furthermore, forcing uniform membrane recruitment of Ena/VASP in developing amacrine cells phenocopied loss of Fat3. Together these results suggest Fat3 polarizes the activity of cytoskeletal effectors to help direct amacrine cell migration and dendrite placement.Medical Science

Mechanical Stimuli Modulate Lateral Root Organogenesis1[W][OA]

Unlike mammals, whose development is limited to a short temporal window, plants produce organs de novo throughout their lifetime in order to adapt their architecture to the prevailing environmental conditions. The production of lateral roots represents one example of such postembryonic organogenesis. An endogenous developmental program likely imposes an ordered arrangement on the position of new lateral roots. However, environmental stimuli such as nutrient levels also affect the patterning of lateral root production. In addition, we have found that mechanical forces can act as one of the triggers that entrain lateral root production to the environment of the Arabidopsis (Arabidopsis thaliana) plant. We observed that physical bending of the root recruited new lateral root formation to the convex side of the resultant bend. Transient bending of 20 s was sufficient to elicit this developmental program. Such bending triggered a Ca2+ transient within the pericycle, and blocking this change in Ca2+ also blocked recruitment of new lateral root production to the curved region of the root. The initial establishment of the mechanically induced lateral root primordium was independent of an auxin supply from the shoot and was not disrupted by mutants in a suite of auxin transporters and receptor/response elements. These results suggest that Ca2+ may be acting to translate the mechanical forces inherent in growth to a developmental response in roots

Two eARCHT3.0 Lines for Optogenetic Silencing of Dopaminergic and Serotonergic Neurons

Dopaminergic and serotonergic neurons modulate and control processes ranging from reward signaling to regulation of motor outputs. Further, dysfunction of these neurons is involved in both degenerative and psychiatric disorders. Elucidating the roles of these neurons has been greatly facilitated by bacterial artificial chromosome (BAC) transgenic mouse lines expressing channelrhodopsin to readily enable cell-type specific activation. However, corresponding lines to silence these monoaminergic neurons have been lacking. We have generated two BAC transgenic mouse lines expressing the outward proton pump, enhanced ArchT3.0 (eArchT3.0), and GFP under control of the regulatory elements of either the dopamine transporter (DAT; Jax# 031663) or the tryptophan hydroxylase 2 (TPH2; Jax# 031662) gene locus. We demonstrate highly faithful and specific expression of these lines in dopaminergic and serotonergic neurons respectively. Additionally we validate effective and sensitive eArchT3.0-mediated silencing of these neurons using slice electrophysiology as well as with a well-established behavioral assay. These new transgenic tools will help expedite the study of dopaminergic and serotonergic system function in normal behavior and disease.National Institute of Neurological Disorders and Stroke (Grants R01NS098505 and R21NS079992)Simons Foundation (Grant 307913)National Institute of Mental Health (Grant R01MH099647)National Institute on Drug Abuse (Grant P50DA042012)Defense Sciences Office (Contract W911NF-14-2-0013

Synthesis, Thermodynamic Properties, and Crystal Structure of RNA Oligonucleotides Containing 5-Hydroxymethylcytosine

International audienc

Denosumab treatment is associated with the absence of circulating tumor cells in patients with breast cancer

Abstract Background The presence of circulating tumor cells (CTCs) in patients with breast cancer correlates to a bad prognosis. Yet, CTCs are detectable in only a minority of patients with progressive breast cancer, and factors that influence the abundance of CTCs remain elusive. Methods We conducted CTC isolation and enumeration in a selected group of 73 consecutive patients characterized by progressive invasive breast cancer, high tumor load and treatment discontinuation at the time of CTC isolation. CTCs were quantified with the Parsortix microfluidic device. Clinicopathological variables, blood counts at the time of CTC isolation and detailed treatment history prior to blood sampling were evaluated for each patient. Results Among 73 patients, we detected at least one CTC per 7.5 ml of blood in 34 (46%). Of these, 22 (65%) had single CTCs only, whereas 12 (35%) featured both single CTCs and CTC clusters. Treatment with the monoclonal antibody denosumab correlated with the absence of CTCs, both when considering all patients and when considering only those with bone metastasis. We also found that low red blood cell count was associated with the presence of CTCs, whereas high CA 15-3 tumor marker, high mean corpuscular volume, high white blood cell count and high mean platelet volume associated specifically with CTC clusters. Conclusions In addition to blood count correlatives to single and clustered CTCs, we found that denosumab treatment associates with most patients lacking CTCs from their peripheral circulation. Prospective studies will be needed to validate the involvement of denosumab in the prevention of CTC generation

Transcriptional and posttranscriptional silencing are mechanistically related

Two distinct gene-silencing phenomena are observed in plants: transcriptional gene silencing (TGS), which involves decreased RNA synthesis because of promoter methylation, and posttranscriptional gene silencing (PTGS), which involves sequence-specific RNA degradation. PTGS is induced by deliberate [1-4] or fortuitous production (R.v.B., unpublished data) of double-stranded RNA (dsRNA). TGS could be the result of DNA pairing [5], but could also be the result of dsRNA, as was shown by the dsRNA-induced inactivation of a transgenic promoter [6]. Here, we show that when targeting flower pigmentation genes in Petunia, transgenes expressing dsRNA can induce PTGS when coding sequences are used and TGS when promoter sequences are taken. For both types of silencing, small RNA species are found, which are thought to be dsRNA decay products [7] and determine the sequence specificity of the silencing process [8, 9]. Furthermore, silencing is accompanied by the methylation of DNA sequences that are homologous to dsRNA. DNA methylation is assumed to be essential for regulating TGS and important for reinforcing PTGS [10]. Therefore, we conclude that TGS and PTGS are mechanistically related. In addition, we show that dsRNA-induced TGS provides an efficient tool to generate gene knockouts, because not only does the TGS of a PTGS-inducing transgene fully revert the PTGS phenotype, but also an endogenous gene can be transcriptionally silenced by dsRNA corresponding to its promoter