Specific and heritable genetic interference by double-stranded RNA in Arabidopsis thaliana

We investigated the potential of double-stranded RNA interference (RNAi) with gene activity in Arabidopsis thaliana. To construct transformation vectors that produce RNAs capable of duplex formation, gene-specific sequences in the sense and antisense orientations were linked and placed under the control of a strong viral promoter. When introduced into the genome of A. thaliana by Agrobacterium-mediated transformation, double-stranded RNA-expressing constructs corresponding to four genes, AGAMOUS (AG), CLAVATA3, APETALA1, and PERIANTHIA, caused specific and heritable genetic interference. The severity of phenotypes varied between transgenic lines. In situ hybridization revealed a correlation between a declining AG mRNA accumulation and increasingly severe phenotypes in AG (RNAi) mutants, suggesting that endogenous mRNA is the target of double-stranded RNA-mediated genetic interference. The ability to generate stably heritable RNAi and the resultant specific phenotypes allows us to selectively reduce gene function in A. thaliana

Engulfing cells promote neuronal regeneration and remove neuronal debris through distinct biochemical functions of CED-1

Two important biological events happen coincidently soon after nerve injury in the peripheral nervous system in C. elegans: removal of axon debris and initiation of axon regeneration. But, it is not known how these two events are co-regulated. Mutants of ced-1, a homolog of Draper and MEGF10, display defects in both events. One model is that those events could be related. But our data suggest that they are actually separable. CED-1 functions in the muscle-type engulfing cells in both events and is enriched in muscle protrusions in close contact with axon debris and regenerating axons. Its two functions occur through distinct biochemical mechanisms; extracellular domain-mediated adhesion for regeneration and extracellular domain binding-induced intracellular domain signaling for debris removal. These studies identify CED-1 in engulfing cells as a receptor in debris removal but as an adhesion molecule in neuronal regeneration, and have important implications for understanding neural circuit repair after injury

Intercellular calcium signaling in a gap junction-coupled cell network establishes asymmetric neuronal fates in C. elegans

The C. elegans left and right AWC olfactory neurons specify asymmetric subtypes, one default AWC(OFF) and one induced AWC(ON), through a stochastic, coordinated cell signaling event. Intercellular communication between AWCs and non-AWC neurons via a NSY-5 gap junction network coordinates AWC asymmetry. However, the nature of intercellular signaling across the network and how individual non-AWC cells in the network influence AWC asymmetry is not known. Here, we demonstrate that intercellular calcium signaling through the NSY-5 gap junction neural network coordinates a precise 1AWC(ON)/1AWC(OFF) decision. We show that NSY-5 gap junctions in C. elegans cells mediate small molecule passage. We expressed vertebrate calcium-buffer proteins in groups of cells in the network to reduce intracellular calcium levels, thereby disrupting intercellular communication. We find that calcium in non-AWC cells of the network promotes the AWC(ON) fate, in contrast to the autonomous role of calcium in AWCs to promote the AWC(OFF) fate. In addition, calcium in specific non-AWCs promotes AWC(ON) side biases through NSY-5 gap junctions. Our results suggest a novel model in which calcium has dual roles within the NSY-5 network: autonomously promoting AWC(OFF) and non-autonomously promoting AWC(ON)

A Toll-interleukin 1 repeat protein at the synapse specifies asymmetric odorant receptor expression via ASK1 MAPKKK signaling

A stochastic lateral signaling interaction between two developing Caenorhabditis elegans AWC olfactory neurons causes them to take on asymmetric patterns of odorant receptor expression, called AWC(OFF) and AWC(ON). Here we show that the AWC lateral signaling gene tir-1 (previously known as nsy-2) encodes a conserved post-synaptic protein that specifies the choice between AWC(OFF) and AWC(ON). Genetic evidence suggests that tir-1 acts downstream of a voltage-gated calcium channel and CaMKII (UNC-43) to regulate AWC asymmetry via the NSY-1(ASK1) p38/JNK MAP (mitogen-activated protein) kinase cascade. TIR-1 localizes NSY-1 to post-synaptic regions of AWC, and TIR-1 binds UNC-43, suggesting that it assembles a synaptic signaling complex that regulates odorant receptor expression. Temperature-shift experiments indicate that tir-1 affects AWC during a critical period late in embryogenesis, near the time of AWC synapse formation. TIR-1 is a multidomain protein with a TIR (Toll-interleukin-1 receptor) domain that activates signaling, SAM repeats that mediate localization to post-synaptic regions of axons, and an N-terminal inhibitory domain. TIR-1 and other TIR proteins are implicated in vertebrate and invertebrate innate immunity, as are NSY-1/ASK1 kinases, so this pathway may also have a conserved function in immune signaling

Analysis of unc-62 expression pattern in C. elegans embryonic AWC neurons

The Caenorhabditis elegans UNC-62 homothorax/Meis/TALE homeodomain protein functions sequentially to regulate general identity of the AWC olfactory neuron pair and the stochastic choice of asymmetric AWC subtypes during embryogenesis. Here we analyze the expression pattern of unc-62 during AWC development using an integrated unc-62::GFP fosmid rescuing transgene. UNC-62::GFP was not detected in AWC neurons in early or late embryos. These results are consistent with previous single-cell RNA sequencing data and also suggest an undetectable level of unc-62 expression and/or low stability of UNC-62 protein in AWC neurons during embryogenesis

mNG-tagged mls-2 knock-in alleles in C. elegans

The Caenorhabditis elegans HMX/NKX MLS-2 transcription factor was previously shown to play sequential roles in AWC general identity and the stochastic AWCON/AWCOFF subtype choice during embryogenesis. Here we analyze the expression pattern of endogenous mls-2 during AWC development using mNeonGreen (mNG) knock-in strains. Similar to transgenic GFP::MLS-2, functional mNG::MLS-2 knock-in displayed nuclear localization in AWC precursor cells but was not observed in AWC during the later embryonic stage. These results suggest that the expression of mls-2 is below the detectable level and/or the stability of MLS-2 protein is very low in AWC cells

CEPsh glia development is not required for general AWC identity or AWC asymmetry

The Caenorhabditis elegans VAB-3/Pax6 homeodomain protein was previously shown to play a role in both the development of cephalic sheath (CEPsh) glia and asymmetric differentiation of AWC olfactory neuron subtypes AWC ON /AWC OFF . Here we show that vab-3 is not required for the specification of general AWC identity. We also show that some vab-3 mutant alleles with defective CEPsh glia development displayed wild-type AWC asymmetry. These results suggest that vab-3 has separable roles in CEPsh glia development and AWC asymmetry. Together, our results suggest that general AWC identity and AWC asymmetry are not dependent on the development of CEPsh glia

The MicroRNA <em>mir-71</em> Inhibits Calcium Signaling by Targeting the TIR-1/Sarm1 Adaptor Protein to Control Stochastic L/R Neuronal Asymmetry in <em>C. elegans</em>

<div><p>The <em>Caenorhabditis elegans</em> left and right AWC olfactory neurons communicate to establish stochastic asymmetric identities, AWC^ON and AWC^OFF, by inhibiting a calcium-mediated signaling pathway in the future AWC^ON cell. NSY-4/claudin-like protein and NSY-5/innexin gap junction protein are the two parallel signals that antagonize the calcium signaling pathway to induce the AWC^ON fate. However, it is not known how the calcium signaling pathway is downregulated by <em>nsy-4</em> and <em>nsy-5</em> in the AWC^ON cell. Here we identify a microRNA, <em>mir-71</em>, that represses the TIR-1/Sarm1 adaptor protein in the calcium signaling pathway to promote the AWC^ON identity. Similar to <em>tir-1</em> loss-of-function mutants, overexpression of <em>mir-71</em> generates two AWC^ON neurons. <em>tir-1</em> expression is downregulated through its 3′ UTR in AWC^ON, in which <em>mir-71</em> is expressed at a higher level than in AWC^OFF. In addition, <em>mir-71</em> is sufficient to inhibit <em>tir-1</em> expression in AWC through the <em>mir-71</em> complementary site in the <em>tir-1</em> 3′ UTR. Our genetic studies suggest that <em>mir-71</em> acts downstream of <em>nsy-4</em> and <em>nsy-5</em> to promote the AWC^ON identity in a cell autonomous manner. Furthermore, the stability of mature <em>mir-71</em> is dependent on <em>nsy-4</em> and <em>nsy-5</em>. Together, these results provide insight into the mechanism by which <em>nsy-4</em> and <em>nsy-5</em> inhibit calcium signaling to establish stochastic asymmetric AWC differentiation.</p> </div

<i>mir-71</i> expression and the <i>tir-1</i> 3′ UTR are differentially regulated in AWC^ON and AWC^OFF neurons.

<p>(A, B) Images of <i>mir-71p::GFP</i>. The AWC^OFF cell body is outlined by dashed lines, which was done when the GFP intensity was temporarily enhanced with the Photoshop levels tool. (A′, B′) Images of <i>ceh-36p::myr-TagRFP</i> and <i>str-2p::2Xnls-TagRFP</i>. AWC^ON was identified as <i>str-2p::2Xnls-TagRFP</i> positive and <i>ceh-36p::myr-TagRFP</i> positive (A′). AWC^OFF was identified as <i>str-2p::2Xnls-TagRFP</i> negative and <i>ceh-36p::myr-TagRFP</i> positive (B′). (A″) Merge of A and A′ images from the same cell. (B″) Merge of B and B′ images from the same cell. (C) Quantification of <i>mir-71p::GFP</i> expression in AWC^ON and AWC^OFF cells. (D, E) Images of <i>odr-3p::GFP::tir-1 3′ UTR</i>. (D′, E′) Images of <i>odr-3p::2Xnls-TagRFP::unc-54 3′ UTR</i> and <i>str-2p::myr-mCherry</i>. The AWC^ON cell was identified as <i>str-2p::myr-mCherry</i> positive and <i>odr-3p::2XTagRFP</i> positive (D′). The AWC^OFF cell was defined as <i>str-2p::myr-mCherry</i> negative and <i>odr-3p::2Xnls-TagRFP</i> positive (E′). (D″) Merge of D and D′ images from the same cell. (E″) Merge of E and E′ images from the same cell. (F) Quantification of normalized GFP expression in AWC^ON and AWC^OFF cells. Normalized GFP expression was determined by calibrating GFP intensity with 2Xnls-TagRFP intensity of the same cell. All constructs, except for <i>odr-3p::GFP::tir-1 3′ UTR</i>, contain the <i>unc-54</i> 3′ UTR. All images were taken from first stage larvae. The single focal plane with the brightest fluorescence in each AWC was selected from the acquired image stack and measured for fluorescence intensity. Each animal was categorized into one of three categories: AWC^ON = AWC^OFF, AWC^ON>AWC^OFF, and AWC^OFF>AWC^ON based on the comparison of GFP intensities between AWC^ON and AWC^OFF cells of the same animal. We did not observe any animals that fell into the “AWC^ON = AWC^OFF” category from our GFP intensity analysis. Total number of animals for each category was tabulated and analyzed as described <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002864#pgen.1002864-Didiano1" target="_blank">[86]</a>. <i>p</i>-values were calculated using <i>X</i>² test. Error bars represent standard error of proportion. Arrows indicate the AWC cell bodies. Arrowheads represent myr-TagRFP or myr-mCherry signal. Scale bar, 2 µm.</p

<i>mir-71</i> acts cell-autonomously to promote AWC^ON.

<p>(A, B) Projections of wild-type animals expressing an integrated <i>str-2p::GFP</i> transgene (green) and an unstable transgenic array containing <i>odr-3p::mir-71</i> and <i>odr-1p::DsRed</i> (red). AWC neurons with co-expression of GFP and DsRed appear yellow. Arrows, AWC cell body; arrowheads, AWB cell body; scale bar, 10 µm. (C, E) AWC phenotypes of wild type (C), <i>nsy-4(ky627)</i>, and <i>nsy-5(ky634)</i> mutants (E) expressing the transgene <i>odr-3p::mir-71; odr-1p::DsRed</i> in both AWC neurons. + and − indicate the presence and absence of the transgene <i>odr-3p::mir-71</i>, respectively. (D, F) AWC phenotypes of wild-type (D) and mutant (F) mosaic animals expressing the transgene <i>odr-3p::mir-71; odr-1p::DsRed</i> in one AWC neuron. Two independent transgenic lines were analyzed in wild type, <i>nsy-4(ky627)</i>, and <i>nsy-5(ky634)</i> mutants in (C–F). Results from two independent lines were similar and thus were combined in (E, F). <i>Z</i>-test was used to calculate <i>p</i> values. (G) Color codes for AWC neurons in (A), (B), (D), and (F).</p