Reevaluation of the role of LIP-1 as an ERK/MPK-1 dual specificity phosphatase in the C. elegans germline

Significance The RAS–ERK pathway is critical for metazoan development. In development, ERK activity is regulated by a balance of phosphorylation of ERK by MEK (MAPK kinase) and dephosphorylation by DUSPs (dual specificity phosphatases). LIP-1, a DUSP6/7 family member, was previously suggested to regulate MPK-1/ERK activity by dephosphorylating MPK-1 in the Caenorhabditis elegans germline, based on LIP-1's mutant phenotype in the germline and its DUSP role in vulval development. However, our investigations demonstrate that LIP-1 does not function as an MPK-1 DUSP in the germline and likely regulates germline functions through distinct targets. Our results present a cautionary note about misinterpreting similar mutant phenotypes caused by mutations in different genes and assuming that genes function similarly in different tissues.


Live imaging of worms
Five to seven animals, each time, were mounted on 2% agarose pads with 10 µl of 0.1 M levamisole in M9 and imaged with Zeiss Axio Imager M2 equipped with an AxioCam MRm camera (Zeiss). All images were obtained using AxioVision software (Zeiss) as a montage of images at 40x with numerical aperture of 0.65.

Progeny assay and embryonic lethality
Ten mid-L4-stage hermaphroditic worms of the indicated genotypes were placed individually on plates. The plates were placed either at 20ºC or shifted to 25ºC, according to the experiment. The parent worm was moved to a fresh plate after every 12 to 18 hours, and the total number of embryos was counted on the original plate. This process was repeated over 5 to 7 days until the worms stopped producing progeny. The total number of embryos (across the entire lay period) was added up for each animal. Similarly, adult progeny was counted from each of those plates after 2-3 days and added for each animal.
Embryonic lethality was calculated in percentage as follows [(number of embryos − number of adults)/numbers of embryos] × 100. The analysis was performed in three to five replicates.

Pachytene progression, oocyte number and Emo phenotype
Dissection and DAPI staining were used for analysis of pachytene progression, oocyte number and Emo phenotype. For each of the experiments, 50-60 mid-L4 worms of the indicated genotypes were placed in a single plate and kept either at 20ºC or shifted to 25ºC for 24 hours. The worms were then dissected and fixed in 3% formaldehyde with 100 mM K2HPO4 (pH 7.2) for 10 min at room temperature washed with phosphate-buffered saline (PBS) and 0.1% Tween 20 (PBST) and post fixed with 100% methanol (−20°C) for 1 hour. The germlines were stained with DAPI (1:1000, stock: 1 µg/mL) for 30 min at roomtemperature and processed for slide preparation. The images were captured with Zeiss (Thornwood, NY) Axio Imager M2 equipped with an AxioCam MRm camera (Zeiss) with Z-stacks (0.6 µm) at 40x with numerical aperture of 0.65. Pachytene germ cells with their distinct morphology were then checked manually in each germline in the proximal gonad (after the loop region). Pachytene-progression defects were scored as the percentage of germlines with any pachytene-stage germ cells located proximal to the loop region.
Oocytes were scored using the DAPI-stained chromosomal morphology of diplotene/diakinesis stage germ cells and counted manually. The Emo phenotype is detected as large DAPI blobs (distinct from 6 bivalents at the diakinesis). Germlines were counted as either with or without Emo phenotype and expressed as percentage.
Staining and dissection was performed as described in main text. Because dpMPK-1 staining was performed in the same tube for both the genotypes, each genotype was first labeled with different markers. fog-2(q71) was labeled with p-SUN-1 (1:800) and lip-1(zh15);fog-2(q71) was labeled with HTP-3 (1:800)]. Wild-type hermaphrodite (mid-L4+24h) germlines were used as a positive control. All the genotypes used in an experiment were then pooled in the same tube for rabbit dpMPK-1 antibody (1:400) and MSP antibody (1:200) incubation for overnight at 4ºC and processed as described (1), followed by secondary antibody staining for anti-Guinea pig (which marks each of the individual genotype), anti-Rabbit for dpMPK-1 and anti-Mouse for detecting MSP, to detect MSP as a marker of successful mating. The slides were made with the pooled immunostained germlines. Wild-type hermaphrodite germlines on each slide were used to set the acquisition time for dpMPK-1 accumulation. Images were acquired with Zeiss Axio Imager M2 equipped with an AxioCam MRm camera (Zeiss). All images were obtained as a montage at 40x with a numerical aperture of 0.65, with overlapping cell boundaries and processed with ImageJ software. The montages were then assembled in Adobe Photoshop CS3 and processed identically.

SYP-1 disassembly assay
For the SYP-1 disassembly assay, 50-60 mid-L4 worms/plate from each genotype were shifted to 25ºC for 24h. The animals were then dissected, and germlines were immunostained with anti-HIM-3 and anti-SYP-1. Because both the antibodies were raised against Rabbit, anti-HIM-3 was first conjugated with Alexa Fluor 488 according to the manufacturer's protocol (APEX TM antibody labeling kit, Thermo Fisher Scientific, A10468).