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1.
Figure 7

Figure 7. Model for mir-71 function in AWC asymmetry.. From: The MicroRNA mir-71 Inhibits Calcium Signaling by Targeting the TIR-1/Sarm1 Adaptor Protein to Control Stochastic L/R Neuronal Asymmetry in C. elegans.

In the default AWCOFF cell, tir-1 acts in a calcium-regulated kinase signaling pathway to represses the expression of the AWCON marker str-2. Both nsy-4 and nsy-5 act to increase the level of mature mir-71, which results in downregulation of tir-1 expression and subsequent de-repression of str-2 gene expression in the cell that becomes AWCON. Gray is used to indicate the gene product is less active or inactive.

Yi-Wen Hsieh, et al. PLoS Genet. 2012 August;8(8):e1002864.
2.
Figure 6

Figure 6. Mature mir-71 level is decreased in nsy-4 and nsy-5 mutants.. From: The MicroRNA mir-71 Inhibits Calcium Signaling by Targeting the TIR-1/Sarm1 Adaptor Protein to Control Stochastic L/R Neuronal Asymmetry in C. elegans.

Stem-loop RT-qPCR analysis of mature and premature mir-71 expression in mir-71(n4115), mir-71(n4115); nsy-5(ky634), and mir-71(n4115); nsy-4(ky627) mutants expressing the odr-3p::mir-71 transgene in AWC. The expression levels of both premature and mature mir-71 were normalized to those of the actin-related gene, arx-1. AU, arbitrary unit. Relative expression was set to one for mir-71(n4115); odr3p::mir-71 and was normalized accordingly for other samples. p values were calculated using Student's t-test. ns, not significant (p = 0.6–0.7). Error bars represent standard error of the mean.

Yi-Wen Hsieh, et al. PLoS Genet. 2012 August;8(8):e1002864.
3.
Figure 3

Figure 3. mir-71 is expressed in AWC.. From: The MicroRNA mir-71 Inhibits Calcium Signaling by Targeting the TIR-1/Sarm1 Adaptor Protein to Control Stochastic L/R Neuronal Asymmetry in C. elegans.

(A, B) Images of a first stage larva expressing the transgenes mir-71p::YFP (A) and odr-1p::DsRed, a marker for AWB and AWC neurons (B). (C) Merged image showing co-expression of YFP and DsRed in AWC and AWB neurons. (D) Quantification of the number of AWC neurons with visible expression of the mir-71p::YFP reporter gene at the first larval stage. Z-test was used to calculate statistical significance. Error bar represents the standard error of proportion. ns, not significant. Arrowhead, AWB cell body; arrow, AWC cell body. Scale bar, 10 µm.

Yi-Wen Hsieh, et al. PLoS Genet. 2012 August;8(8):e1002864.
4.
Figure 5

Figure 5. mir-71 acts cell-autonomously to promote AWCON.. From: The MicroRNA mir-71 Inhibits Calcium Signaling by Targeting the TIR-1/Sarm1 Adaptor Protein to Control Stochastic L/R Neuronal Asymmetry in C. elegans.

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

Yi-Wen Hsieh, et al. PLoS Genet. 2012 August;8(8):e1002864.
5.
Figure 1

Figure 1. mir-71 promotes the AWCON identity.. From: The MicroRNA mir-71 Inhibits Calcium Signaling by Targeting the TIR-1/Sarm1 Adaptor Protein to Control Stochastic L/R Neuronal Asymmetry in C. elegans.

(A–D) Expression of a stable transgene str-2p::GFP (AWCON marker) in wild type (A), tir-1(tm3036) loss-of-function (lf) mutants (B), mir-71(OE) animals overexpressing the transgene odr-3p::mir-71 in AWCs (C), and tir-1(ky648gf) mutants (D). tir-1(ky648gf) mutants also carry the transgene odr-1p::DsRed (expressed in both AWCON and AWCOFF) to show that the absence of str-2p::GFP expression is not due to loss of AWC neurons. (E) str-2p::GFP expression phenotypes in wild type, single mutants, and double mutants. nsy-4(OE) animals overexpress the transgene odr-3p::nsy-4 in AWCs. (F) Genetic map of mir-71. mir-71 (blue arrow) is located in an intron of F16A11.3a encoding the ppfr-1 gene. Black bars indicate the location of deletions in ppfr-1(tm2180) and mir-71(n4115) mutants. A schematic of the GFP reporter gene driven by a 2.4 kb region upstream of mir-71 transcript is shown. Arrows, AWC cell body. Scale bar, 10 µm. Statistical analysis was performed using the Z-test for two proportions: *p<0.05; ***p<0.001; ns, not significant.

Yi-Wen Hsieh, et al. PLoS Genet. 2012 August;8(8):e1002864.
6.
Figure 2

Figure 2. mir-71 downregulates gene expression through the tir-1 3′ UTR.. From: The MicroRNA mir-71 Inhibits Calcium Signaling by Targeting the TIR-1/Sarm1 Adaptor Protein to Control Stochastic L/R Neuronal Asymmetry in C. elegans.

(A) Complementarity between the mir-71 seed region and the tir-1 3′ UTR in C. elegans and C. briggsae. Asterisks denote nucleotides mutated in the predicted mir-71 target site of the tir-1 3′ UTR in (B). (B) Left: GFP sensor constructs, driven by the odr-3 promoter, with the tir-1 3′ UTR or the tir-1 3′ UTR mutated in the predicted mir-71 target site. Middle: Images of GFP expression from GFP sensor constructs and nucleus-localized TagRFP expression from the internal control transgene odr-3p::2Xnls-TagRFP::unc-54 3′ UTR in the AWC cell body of wild type and mir-71(OE) animals. All images were taken from animals in the first larval stage. Scale bar, 5 µm. Arrows, AWC cell body. Right: The average normalized GFP intensity of each sensor construct in the AWC cell body. The GFP intensity of an individual cell was normalized to the TagRFP intensity of the same cell. For each sensor construct line, the normalized GFP intensity in wild type was set as 1 arbitrary unit (AU) and the normalized GFP intensity in mir-71(OE) was calibrated to that in wild type. Student's t-test was used for statistical analysis. n = 16–21 for each transgenic line in wild type and mir-71(OE) animals. Error bars, standard error of the mean. ns, not significant. (C) Left: tir-1 overexpression constructs, driven by the odr-3 promoter, with the tir-1 3′ UTR or the tir-1 3′ UTR mutated in the predicted mir-71 target site. Right: Normalized fold change in tir-1(OE) 2AWCOFF phenotype. The fold change in tir-1(OE) 2AWCOFF phenotype was determined by dividing the 2AWCOFF percentage of tir-1(OE) with the 2AWCOFF percentage of tir-1(OE); mir-71(OE), which was then normalized to the relative tir-1(OE) transgene copy number. Two to three independent lines were analyzed for each tir-1 overexpression construct. Student's t-test was used to calculate statistical significance. Error bars represent standard error of the mean.

Yi-Wen Hsieh, et al. PLoS Genet. 2012 August;8(8):e1002864.
7.
Figure 4

Figure 4. mir-71 expression and the tir-1 3′ UTR are differentially regulated in AWCON and AWCOFF neurons.. From: The MicroRNA mir-71 Inhibits Calcium Signaling by Targeting the TIR-1/Sarm1 Adaptor Protein to Control Stochastic L/R Neuronal Asymmetry in C. elegans.

(A, B) Images of mir-71p::GFP. The AWCOFF 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 ceh-36p::myr-TagRFP and str-2p::2Xnls-TagRFP. AWCON was identified as str-2p::2Xnls-TagRFP positive and ceh-36p::myr-TagRFP positive (A′). AWCOFF was identified as str-2p::2Xnls-TagRFP negative and ceh-36p::myr-TagRFP 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 mir-71p::GFP expression in AWCON and AWCOFF cells. (D, E) Images of odr-3p::GFP::tir-1 3′ UTR. (D′, E′) Images of odr-3p::2Xnls-TagRFP::unc-54 3′ UTR and str-2p::myr-mCherry. The AWCON cell was identified as str-2p::myr-mCherry positive and odr-3p::2XTagRFP positive (D′). The AWCOFF cell was defined as str-2p::myr-mCherry negative and odr-3p::2Xnls-TagRFP 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 AWCON and AWCOFF cells. Normalized GFP expression was determined by calibrating GFP intensity with 2Xnls-TagRFP intensity of the same cell. All constructs, except for odr-3p::GFP::tir-1 3′ UTR, contain the unc-54 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: AWCON = AWCOFF, AWCON>AWCOFF, and AWCOFF>AWCON based on the comparison of GFP intensities between AWCON and AWCOFF cells of the same animal. We did not observe any animals that fell into the “AWCON = AWCOFF” category from our GFP intensity analysis. Total number of animals for each category was tabulated and analyzed as described . p-values were calculated using X2 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.

Yi-Wen Hsieh, et al. PLoS Genet. 2012 August;8(8):e1002864.

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