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. 2021 Dec 15;10:e66955. doi: 10.7554/eLife.66955

Figure 2. che-1 mRNA and protein copy numbers in ASE neurons.

(A) Expression of che-1 and the CHE-1 target gcy-22 in embryos at the bean (top) and 2-fold stage (bottom). Each spot is a single mRNA molecule visualized by single molecule FISH (smFISH). Dashed lines outline ASE neuron cell bodies. Scale bar: 2 μm. (B) che-1 (green) and gcy-22 (red) mRNA levels during embryonic development. che-1 expression peaks during specification, but falls as expression of CHE-1 target genes rise. (C) Expression of che-1 and CHE-1 targets gcy-22, gcy-14, tax-2 and del-2 visualized by smFISH in L2 larvae. Dashed lines outline left and right ASE neurons (ASEL/R). Scalebar: 2 μm. (D) Quantification of expression of che-1 and CHE-1 targets in ASEL (L) and ASER (R) in L2 larvae. che-1 mRNA levels are low compared to other CHE-1 target genes. (E) Low che-1 expression (green, ASER/L) compared to expression of CHE-1 targets gcy-22 (red, ASER) and gcy-14 (red, ASEL) throughout larval development. Body length corresponds to developmental time, with approximate timing of hatching and molts between larval stages L1-L4 indicated by vertical lines. (F) Left panel: two-photon microscopy image of L2 larva expressing endogenously-tagged CHE-1::GFP, immersed in 72 nM eGFP for calibration. Right panel: CHE-1::GFP protein molecules in ASER (green), (R) and ASEL (blue), (L) at different stages of post-embryonic development. The number of CHE-1 proteins is comparable to the predicted number of CHE-1-binding sites. Error bars in B, D, and F represent mean ± SD. ***p < 0.001.

Figure 2—source data 1. Data and scripts for Figure 2 and related figure supplements.

Figure 2.

Figure 2—figure supplement 1. CHE-1 copy numbers in larvae and embryos.

Figure 2—figure supplement 1.

(A) CHE-1::GFP protein concentration in ASER (green) and ASEL (blue) at different stages of post-embryonic development (L1/L2: circles, L3/L4: triangle, young adult: squares). CHE-1::GFP protein concentration decreases with the age of the animals. (B) Images of che-1::GFP embryos for quantification of CHE-1::GFP in ASE neurons with confocal microscopy (left). Number of CHE-1::GFP protein molecules over time in embryos, starting at the bean stage until twitching started (at 22 °C) (right). Embryos showed similar levels of CHE-1::GFP protein as the L1 larvae. (C) che-1::GFP::AID mRNA copy number under normal conditions (wild-type animals, no EtOH) and in presence of EtOH (che-1::GFP::AID animals without auxin but with 0.25% EtOH). As auxin is dissolved in 100% EtOH, auxin treatments occurred at ~0.25% EtOH. This control indicates that mRNA levels are not changed due to the presence of EtOH during auxin treatment. L/R indicates ASEL/R.