Single-sister labeling in the C. elegans germline using the nucleotide analog EdU

Abstract

Reciprocal exchanges between genetically identical sister chromatids (sister chromatid exchanges or SCEs) have been challenging to study. Here, we describe a protocol that utilizes a pulse/chase of the thymidine analog 5-ethyl-3'-deoxyuridine (EdU) in combination with click chemistry and antibody labeling to selectively label sister chromatids in the C. elegans germline. Labeling has no discernable effects on meiosis, allowing for cytological quantification of SCEs. This protocol can be combined with a variety of imaging approaches, including STED, confocal and super-resolution. For complete details on the use and execution of this protocol, please refer to Almanzar et al. (2021).

Keywords: Cell Biology; Genetics; Microscopy; Model Organisms; Molecular Biology; Molecular/Chemical Probes.

Graphical abstract

Figure 1

EdU incorporation into C. elegans (A) Pipetting M9/Triton X onto a plate of L4 worms. (B) Aspirating M9/Triton X with L4 worms. (C) Transferring M9/Triton X and L4 worms to a fresh 1.5 mL tube. (D) Spinning down worms. (E) Washing once with M9/Triton X. (F) Aspirating 40 μL of M9/Triton containing the worms into a fresh 1.5 mL tube. (G) Adding 60 μL of 10 mM EdU. (H) Vortexing gently to mix worms and EdU. (I) Placing worms and EdU onto a nutator.

Figure 2

Dissection and Fixation of C. elegans gonads (A) Placing a 22 × 22 mm coverslip onto a transparent glass slide on a dissecting stereo microscope. (B) Attaching a fresh blade to a scalpel handle (Caution - blades are very sharp!). (C) Adding EBT solution to the center of the coverslip. (D) Transferring worms by picking into the EBT solution. (E) Dissecting C. elegans gonads by using the scalpel to remove the head or tail of the animal. (F) Adding fix solution to the worms in EBT. (G) Aspirating as much liquid as possible (∼45 μL), being careful not to aspirate the worms. (H) Gently touching the bonded side of the slide to the coverslip - the coverslip and worms should stick to the Histobond slide. (I) Transferring the slide, coverslip facing up, onto an ice block cooled on dry ice. (J) Using a razor blade, flicking off the coverslip by inserting the blade under a corner and gently prying. (K) Transferring the slide into 100% methanol cooled to −20°C. (L) Transferring slides to a fresh Coplin jar of PBST.

Figure 3

Antibody staining of C. elegans gonads (A) Blocking slides in 1× Roche Block. (B) Adding 100 μL of primary antibody solution to a parafilm square (one square per slide). (C) Removing slide from PBST and gently drying the back of the slide, taking care not to disturb the worms stuck to the front of the slide. (D) Preparing the humid chamber by wetting a paper towel and placing it on the bottom of a perforated chamber with a lid. (E) Gently touching the slide (worms down) to the primary antibody. The parafilm should stick to the slide. (F) Incubating the slides, parafilm side up, in a humid chamber with the lid closed. (G) Transferring slides to a fresh, fully filled Coplin jar of PBST, allowing the parafilm squares to float to the top. Steps depicted in panels (B–G) are repeated for the secondary antibody labeling and click chemistry steps.

Figure 4

Mounting and sealing (A) Adding 1 drop (∼12 μL) of Prolong Glass or NPG-Glycerol to a fresh coverslip. (B) Gently touching the samples to the mounting media on the coverslip. (C) If using NPG-Glycerol, aspirating excess mounting media using a pipette tip connected to a vacuum trap. (D) Sealing the slides with nail polish. (E) Storing slides covered at 4°C or −20°C before imaging.

Figure 5

Imaging C. elegans gonads stained for EdU (A) Placing slide coverslip down onto a dissecting scope, between two clear slides with tape. (B) Locating the worm carcasses (black arrows) and marking them with a nearby dot. (C) Mounting slide coverslip down onto your imaging platform. Use the black dots to help locate the sample and image.

Figure 6

Single sister labeling in C. elegans chromosomes Chromosome axis (HTP-3) is labeled in red, EdU is in green and DAPI in blue. (A) A bivalent chromosome with two EdU containing strands (one per sister pair). (B) Two univalent chromosome with two EdU containing sisters (one per sister pair). Unlabeled autosome is marked with an asterisk. (C) An EdU containing sister that underwent an SCE (white arrow) in a bivalent chromosome. (D) A univalent chromosome with an EdU containing sister that underwent an SCE (white arrow). Scale bars = 1 μm. Reprinted with permission from (Almanzar et al., 2021).

Figure 7

EdU incorporation patterns in the C. elegans gonad EdU can be incorporated into only the X chromosome (yellow circles), all six chromosomes (green circles), or only the autosomes (red circles), depending on the time when EdU is present during S phase. The nuclei are circled using the same color scheme to indicate their relative location in the gonad. Bottom, a gonad dissected after five hours of chase at 20°C from an N2 animal labeled as a young adult.

Figure 8

Single sister labeling in diakinesis Gonad zones are labeled: PMZ, premeiotic zone; TZ, transition zone; EP, Early Pachytene, MP, Mid-Pachytene, LP, Late Pachytene, Diplotene, and Diakinesis (square). Inset, magnification of the -2, -3, -4, and -5 nuclei indicate all are successfully labeled with EdU. Numbering is indicative of the position relative to the fertilized oocyte, i.e., -3 is two nuclei before the terminal diakinesis nucleus. Note that each nucleus contains a different number of EdU labeled chromosomes. Axis (HTP-3) is in red (omitted in the zoomed-out image for clarity), EdU is in green and DAPI in blue.

Figure 9

Schematic of pulse/chase experiment Each vertical section represents an additional S phase (mitotic or pre-meiotic). Top, EdU incorporation results in all sisters containing EdU. Middle, following an additional S phase without EdU reduces the number of EdU containing sisters to two out of four per chromosome, allowing for quantification of reciprocal exchanges between labeled and unlabeled chromatin. Bottom, an additional S phase without EdU before meiosis further reduce the number of EdU containing sisters.

Figure 10

EdU progression after various of chase times (A–C) N2 worms incorporated EdU as L4 and dissected at 24-, 27-, and 30-h after EdU incorporation. (D–F) Worms incorporated EdU as young adults (24 h post-L4) and dissected at 24-, 27- and 30-h after EdU incorporation. Note the progression of EdU signal in the gonad, and the dramatically more proximal signal in worms incorporating EdU as L4s versus young adults.

Figure 11

Quantification of EdU progression after various of chase times Distribution of EdU containing nuclei in L4 and young adult N2 and him-8 worms at different times of dissection. The furthest EdU labeled nucleus in each gonad was used to define the stage in meiotic prophase reached by nuclei. The y axis represents percentage of total gonads per experimental group that were labeled in that region. Note the more proximal movement in L4 relative to the young adults in both the N2 and him-8 backgrounds. At least 6 gonads were analyzed in each timepoint.