Stress-induced changes to the dendritic architecture of neurons have been demonstrated in numerous mammalian and invertebrate systems. Remodeling of dendrites varies tremendously among neuron types. During the stress-induced dauer stage of Caenorhabditis elegans, the IL2 neurons arborize to cover the anterior body wall. In contrast, the FLP neurons arborize to cover an identical receptive field during reproductive development. Using timecourse imaging, we show that branching between these two neuron types is highly coordinated. Furthermore, we find that the IL2 and FLP arbors have a similar dendritic architecture and use an identical downstream effector complex to control branching; however, regulation of this complex differs between stress-induced IL2 branching and FLP branching during reproductive development. We demonstrate that the unfolded protein response (UPR) sensor IRE-1, required for localization of the complex in FLP branching, is dispensable for IL2 branching at standard cultivation temperatures. Exposure of ire-1 mutants to elevated temperatures results in defective IL2 branching, thereby demonstrating a previously unknown genotype by environment interaction within the UPR. We find that the FOXO homolog, DAF-16, is required cell-autonomously to control arborization during stress-induced arborization. Likewise, several aspects of the dauer formation pathway are necessary for the neuron to remodel, including the phosphatase PTEN/DAF-18 and Cytochrome P450/DAF-9. Finally, we find that the TOR associated protein, RAPTOR/DAF-15 regulates mutually exclusive branching of the IL2 and FLP dendrites. DAF-15 promotes IL2 branching during dauer and inhibits precocious FLP growth. Together, our results shed light on molecular processes that regulate stress-mediated remodeling of dendrites across neuron classes.
Edited by Ned ManteiKeywords: RNAi Caenorhabditis elegans males Non-disjunction Kinesin-like protein Him phenotype dsRNA a b s t r a c t Rare Caenorhabditis elegans males arise when sex chromosome non-disjunction occurs during meiosis in self-fertilizing hermaphrodites. Non-disjunction is a relatively rare event, and males are typically observed at a frequency of less than one in five hundred wild-type animals. Males are required for genetic crosses and phenotypic analysis, yet current methods to generate large numbers of males can be cumbersome. Here, we identify RNAi reagents (dsRNA-expressing bacteria) with improved effectiveness for eliciting males. Specifically, we used RNAi to systematically reduce the expression of over two hundred genes with meiotic chromosome segregation functions, and we identified a set of RNAi reagents that robustly and reproducibly elicited male progeny.
Stress influences the shape of dendritic arbors in neurons. During the stress-induced dauer stage of Caenorhabditis elegans, the IL2 neurons arborize to cover the anterior body wall. In contrast, the FLP neurons arborize to cover the anterior body wall during non-dauer development.Previous work showed that the membrane-bound receptor DMA-1 regulates FLP branching as part of a larger protein complex. Using forward genetics, we show that the IL2 neurons also use the DMA-1 complex to regulate branching. To understand the coordination of the IL2s and FLPs we conducted a time-course examination of FLPs and found previously undescribed branching patterns indicating a neighborhood effect wherein the FLPs and IL2s in the anterior have differential branching compared to the more posteriorly located PVD arborizing neurons. To determine how the IL2s and FLPs differentially regulate branching, we examined several regulators of DMA-1 localization. We show that the unfolded protein response sensor IRE-1, required for FLP branching, is only required for dauer-specific branching at elevated temperatures. Interestingly, we found that ire-1 mutants have broad, organism-wide temperaturedependent effects on dauer remodeling, suggesting a previously undescribed role for IRE-1 in phenotypic plasticity. We also found that defects in other regulators of dauer remodeling including DAF-16/FOXO, DAF-9/Cytochrome P450, and DAF-18/PTEN are required for proper IL2 arborization, but dispensable for FLP branching. Interestingly, we find that TOR adaptor protein DAF-15/RAPTOR is both required for promoting IL2 branching and inhibiting precocious development of the FLPs. Our results demonstrate specific genotypic by environmental interactions regulating dendrite arborization.
Organisms used as model genomics systems are maintained as isogenic strains, yet evidence of sequence differences between independently maintained wild-type stocks has been substantiated by whole-genome resequencing data and strain-specific phenotypes. Sequence differences may arise from replication errors, transposon mobilization, meiotic gene conversion, or environmental or chemical assault on the genome. Low frequency alleles or mutations with modest effects on phenotypes can contribute to natural variation, and it has proven possible for such sequences to become fixed by adapted evolutionary enrichment and identified by resequencing. Our objective was to identify and analyze single locus genetic defects leading to RNAi resistance in isogenic strains of Caenorhabditis elegans. In so doing, we uncovered a mutation that arose de novo in an existing strain, which initially frustrated our phenotypic analysis. We also report experimental, environmental, and genetic conditions that can complicate phenotypic analysis of RNAi pathway defects. These observations highlight the potential for unanticipated mutations, coupled with genetic and environmental phenomena, to enhance or suppress the effects of known mutations and cause variation between wild-type strains.
We describe a workflow for efficient, environmentally attentive, and sustainable practices related to routine agarose gel electrophoresis. The methods reduce plastic waste and improve efficiency, especially for the exhaustive screening of difficult-to-obtain plasmids. Sustainability is increased when agarose is used ten times over by virtue of a thorough recycling regimen. The workflow optimizes workspaces and standardizes lab practices for handling potentially hazardous waste, minimizing environmental harm. Safety, efficiency, and sustainability improve laboratory productivity, help minimize environmental contamination, and increase cost-effectiveness.
A common laboratory method involves gel electrophoresis followed by photographic documentation of the results, a procedure which is performed worldwide by students and experienced scientists alike. Proprietary Gel Documentation Systems are convenient and useful for documentation of electrophoresis results, but the systems can be prohibitively expensive to purchase and repair, they contain features that are not necessary for everyday documentation, and some users may not find the systems intuitive to operate. We describe our gel documentation setup that meets the everyday needs for documentation in our lab. The setup is inexpensive, modular, user friendly, and increases sustainability through extending the working life of obsolete cell phones, iPads, or other electronic devices containing a camera. More importantly, the setup completely shields users from potentially damaging ultraviolet radiation.
Journal of Cardiovascular Magnetic Resonance 2009, 11(Suppl 1):O1Introduction: Prophylactic implantation of a cardioverter/ defibrillator (ICD) has been shown to reduce mortality in patients with chronic myocardial infarction (CMI) and an increased risk for life threatening ventricular arrhythmia (VA). The use of ICDs in this large patient population is still limited by high costs and possible adverse events including inappropriate discharges and progression of heart failure. VA is related to infarct size and seems to be related to infarct morphology. Contrast enhanced cardiovascular magnetic resonance imaging (ceCMR) can detect and quantify myocardial fibrosis in the setting of CMI and might therefore be a valuable tool for a more accurate risk stratification in this setting. Hypothesis: ceCMR can identify the subgroup developing VA in patients with prophylactic ICD implantation following MADIT criteria. Methods: We prospectively enrolled 52 patients (49 males, age 69 ± 10 years) with CMI and clinical indication for ICD therapy following MADIT criteria. Prior to implantation (36 ± 78 days) patients were investigated on a 1.5 T clinical scanner (Siemens Avanto © , Germany) to assess left ventricular function (LVEF), LV end-diastolic volume (LVEDV) and LV mass (sequence parameters: GRE SSFP, matrix 256 × 192, short axis stack; full LV coverage, no gap; slice thickness 6 mm). For quantitative assessment of infarct morphology late gadolinium enhancement (LGE) was performed including measurement of total and relative infarct mass (related to LV mass) and the degree of transmurality (DT) as defined by the percentage of transmurality in each scar. (sequence parameters: inversion recovery gradient echo; matrix 256 × 148, imaging 10 min after 0.2 μg/kg gadolinium DTPA; slice orientation equal to SSFP). MRI images were analysed using dedicated software (MASS © , Medis,
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