Conditional immobilization for live imaging Caenorhabditis elegans using auxin-dependent protein depletion

Abstract

The visualization of biological processes using fluorescent proteins and dyes in living organisms has enabled numerous scientific discoveries. The nematode Caenorhabditis elegans is a widely used model organism for live imaging studies since the transparent nature of the worm enables imaging of nearly all tissues within a whole, intact animal. While current techniques are optimized to enable the immobilization of hermaphrodite worms for live imaging, many of these approaches fail to successfully restrain the smaller male worms. To enable live imaging of worms of both sexes, we developed a new genetic, conditional immobilization tool that uses the auxin-inducible degron (AID) system to immobilize both adult and larval hermaphrodite and male worms for live imaging. Based on chromosome location, mutant phenotype, and predicted germline consequence, we identified and AID-tagged three candidate genes (unc-18, unc-104, and unc-52). Strains with these AID-tagged genes were placed on auxin and tested for mobility and germline defects. Among the candidate genes, auxin-mediated depletion of UNC-18 caused significant immobilization of both hermaphrodite and male worms that was also partially reversible upon removal from auxin. Notably, we found that male worms require a higher concentration of auxin for a similar amount of immobilization as hermaphrodites, thereby suggesting a potential sex-specific difference in auxin absorption and/or processing. In both males and hermaphrodites, depletion of UNC-18 did not largely alter fertility, germline progression, nor meiotic recombination. Finally, we demonstrate that this new genetic tool can successfully immobilize both sexes enabling live imaging studies of sexually dimorphic features in C. elegans.

Keywords: C. elegans; auxin-inducible degron system; gametogenesis; germ cell development; germline; live imaging; meiosis; oogenesis; spermatogenesis; worms.

Figure 1

Auxin-dependent depletion of unc-18 and unc-104 permits conditional immobilization of hermaphrodites. (A) Diagram of the AID system showing how TIR1 associates with the endogenous SCF E3 ligase complex that in the presence of auxin cause ubiquination (Ub) of the AID*::YFG-1 protein (Your Favorite Gene 1). This ubiquination results in proteosomal degradation of AID*::YFG-1. (B) Schematics of the three candidate genes CRISPR/Cas9 tagged with AID* and the TIR1 constructs used with the expression location of TIR1 based on the eft-3 or rgef-1 promoters. The chromosome number where each construct is located in the genome is indicated on the left of each schematic. (C) Quantification of the average speed in pixels per second of wild type (n = 30), unc-18::AID*; rgef-1p::TIR1 (no auxin n = 18, 1 mM auxin n = 16, recovery n = 26), rgef-1p::TIR1; unc-104::AID* (no auxin n = 24, 1 mM auxin n = 31, recovery n = 18), and eft-3p::TIR1; unc-104::AID* (no auxin n = 15, 1 mM auxin n = 26, recovery n = 24) on plates containing no auxin and 1 mM auxin. The recovery category indicates worms that have been off auxin for 18–24 h prior to tracking worm motion. **** indicates P < 0.00001 and *** indicates P < 0.0001, Kruskal–Wallis/Dunn’s multiple comparisons.

Figure 2

Auxin-dependent depletion of UNC-18 does not significantly alter fertility. (A) Brood size calculations and (B) progeny counts of lives worms, dead eggs, and unfertilized (unfert.) eggs for wild type, unc-18::AID*; rgef-1p::TIR1, rgef-1p::TIR1; unc-104::AID*, and eft-3p::TIR1; unc-104::AID* on NGM with and without 1 mM auxin. N-value indicates the number of parental hermaphrodites scored and bagged/missing indicates the number of worms that bagged or disappeared from the plate during the 5 days of scoring. * indicates P < 0.01, ** indicates P < 0.001, *** indicates P < 0.0001, Dunnett’s multiple comparisons test.

Figure 3

Hermaphrodite germline progression and meiotic crossover formation are unaffected by auxin-depletion of unc-18. (A) Quantification of the nuclear progression through the germline in wild type with no auxin exposure (grey) and unc-18::AID*; rgef-1p::TIR1 on 1 mM auxin plates (green). Each gonad was scored based on the germline position of the last EdU stained nucleus from gonads dissected at 0, 10, 24, and 48 h post-EdU labeling. For this analysis, the germline (diagrammed on the left) was divided into four regions: premeiotic tip to transition zone (PMT-TZ), early pachytene to mid pachytene (EP-MP), mid pachytene to late pachytene (MP-LP), and diakinesis to end of the germline (Diakinesis+). N-values for the number of worms scored are displayed on the plot below each bar. (B) Representative images of dissected gonads from wild type (no auxin exposure) and unc-18::AID*; rgef-1p::TIR1 (1 mM auxin exposure) hermaphrodites stained for RAD-51 (green) and DNA (DAPI, blue). Scale bar represents 20 µm and white line indicates the length of the RAD-51 staining within the germline. (C) Quantification of the number of RAD-51 foci per nucleus for wild type (grey, no auxin exposure, n = 6 gonads, 791 TZ nuclei, 352 EP nuclei, 284 MP nuclei, 253 LP nuclei) and unc-18::AID*; rgef-1p::TIR1 (green, 1 mM auxin exposure, n = 6 gonads, 615 TZ nuclei, 422 EP nuclei, 261 MP nuclei, 224 LP nuclei). Statistics determined using Kruskal–Wallis/Dunn’s multiple comparisons with * representing the number of significant digits after the decimal and “ns” meaning not significant. (D) Representative image and quantification of diakinesis chromosomes (DAPI staining bodies) from wild type (no auxin exposure, n = 30 oocytes) and unc-18::AID*; rgef-1p::TIR1 (1 mM auxin exposure, n = 30 oocytes).

Figure 4

Conditional immobilization of hermaphrodites for live imaging. (A) Brightfield timelapse montage of an immobilized hermaphrodite worm at 40X magnification with images captured every 90 s for 60 min. The montage displays every third frame of the timelapse. Yellow arrowhead indicates ovulation of an egg by the immobilized hermaphrodite. (B) Autofluorescence from an immobilized L1 worm at 60X magnification with images captured every 2 min for 60 min. The montage displays every third frame of the timelapse. (C) mCherry::H2B in the germline of an immobilized L4 worm captured at 60X magnification with images captured every 2 min for 60 min. The montage displays every second frame of the timelapse for 44 min. Yellow arrowheads identify nuclei in the frame undergoing mitotic divisions. (D) SYP-2::GFP timelapse montage of the hermaphrodite germline at 60X magnification with images captured every 5 min for 65 min. The mid-pachytene region of the germline is shown and germline is moving from left to right in each image. The yellow-dashed box indicates the nucleus that is enlarged in the inset panel (yellow outline) with the scale bar in the inset panel representing 2 µm. The complete movies can be viewed in Supplementary Movies S1–S5.

Figure 5

Conditional immobilization of males following auxin-dependent depletion of unc-18. (A) Quantification of the average speed in pixels per second of wild type (n = 24) and unc-18::AID*; rgef-1p::TIR1 on plates containing no auxin (n = 29), 1 mM auxin (n = 25), recovery from 1 mM auxin (n = 21), 10 mM auxin (n = 30), and recovery from 10 mM auxin (n = 21). The recovery category indicates worms that have been off auxin for 18–24 h prior to tracking worm motion. *** indicates P < 0.0001 and **** indicates P < 0.00001, Kruskal–Wallis/Dunn’s multiple comparisons. (B) Quantification of the nuclear progression through the germline in wild type with no auxin exposure and on 10-mM auxin plates and unc-18::AID*; rgef-1p::TIR1 on 10-mM auxin plates. Each gonad was scored based on the germline position of the last EdU stained nucleus from gonads dissected at 0, 10, and 24 h post-EdU labeling. For this analysis, the germline (diagrammed on the left) was divided into four regions: premeiotic tip to transition zone (PMT-TZ), early pachytene to mid pachytene (EP-MP), mid pachytene to late pachytene (MP-LP), and diakinesis to end of the germline (Diakinesis+). N-values for the number of worms scored are displayed on the plot below each bar. (C) Representative images of dissected gonads from wild type (no auxin exposure) and unc-18::AID*; rgef-1p::TIR1 (1 mM auxin exposure) males stained for RAD-51 (green) and DNA (DAPI, blue). The scale bar represents 20 µm and white line indicates the length of the RAD-51 staining within the germline. (D) Quantification of the number of RAD-51 foci per nucleus for wild type (grey, no auxin exposure, n = 6 gonads, 206 TZ nuclei, 151 EP nuclei, 100 MP nuclei, and 127 LP nuclei) and unc-18::AID*; rgef-1p::TIR1 (green, 10 mM auxin exposure, n = 6 gonads, 221 TZ nuclei, 115 EP nuclei, 94 MP nuclei, and 143 LP nuclei). Statistics determined using Kruskal–Wallis/Dunn’s multiple comparisons with * representing the number of significant digits after the decimal and “ns” meaning not significant.

Figure 6

Conditional immobilization of males for live imaging. (A) Brightfield timelapse montage of an immobilized male worm at 20X magnification with images captured every 90 s for 60 min. The montage displays every third frame of the timelapse. (B) SYP-2::GFP timelapse montage of the male germline at 60X magnification with images captured every 5 min for 65 min. The mid-pachytene region of the germline is shown and germline is moving from bottom left to top right in each image. The yellow-dashed box indicates the nucleus that is enlarged in the inset panel (yellow outline) with the scale bar in the inset panel representing 2 µm. The complete movies can be viewed in Supplementary Movies S6–S8.