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Data from: Caenorhabditis elegans germ granules accumulate hundreds of low translation mRNAs with no systematic preference for germ cell fate regulators

Cite this dataset

Scholl, Alyshia (2024). Data from: Caenorhabditis elegans germ granules accumulate hundreds of low translation mRNAs with no systematic preference for germ cell fate regulators [Dataset]. Dryad. https://doi.org/10.5061/dryad.02v6wwq98

Abstract

In animals with germ plasm, embryonic germline precursors inherit germ granules, condensates proposed to regulate mRNAs coding for germ cell fate determinants. In C. elegans, mRNAs are recruited to germ granules by MEG-3, a sequence non-specific RNA-binding protein that forms stabilizing interfacial clusters on germ granules. Using fluorescent in situ hybridization, we confirmed that 441 MEG-3-bound transcripts distribute in a pattern consistent with enrichment in germ granules. 13 are related to transcripts reported in germ granules in Drosophila or Nasonia. The majority, however, are low-translation maternal transcripts required for embryogenesis that are not maintained preferentially in the nascent germline. Granule enrichment raises the concentration of certain transcripts in germ plasm but is not essential to regulate mRNA translation or stability. Our findings suggest that only a minority of germ granule-associated transcripts contribute to germ cell fate in C. elegans and that the vast majority function as non-specific scaffolds for MEG-3.

README: Caenorhabditis elegans germ granules accumulate hundreds of low translation mRNAs with no systematic preference for germ cell fate regulators

https://doi.org/10.5061/dryad.02v6wwq98

in situ screen of MEG-3-bound RNAs identified in Lee et al., 2020. Images were collected across 7 stages (2-cell, 4-cell, 8-cell, 28-cell, ~40-cell, ~60-cell, ~100-cell). 2-cell, 4-cell, 8-cell and 28-cell embryos were scored manually for granules visible in P1, P2, P3, and P4 blastomeres (P blastomeres are readily identified at these stages based on their position in the embryo). Transcripts positive for granules were classified as Group I or II, and transcripts negative for granules were classified as Group III. 

The distinction between Group I and Group II transcripts was made by examining ~40-cell, ~60-cell, ~100-cell stage embryos. Transcripts that showed consistent enrichment in one cell (40-cell and 60-cell stages) and two cells (100-cell stage) across these stages were classified as Group I. Because we relied on position in the embryo, rather than a marker, to identify P4 and the PGCs in these stages, we can only state that Group I transcripts exhibit patterns consistent with preferential maintenance in the P lineage to the PGC stage. Transcripts that lacked obvious enrichment at the 100-cell stage were classified as Group II (transcripts not enriched in PGCs). Five Group III transcripts (vdac-1, gld-1, F22D6.2, dlc-1, *and *act-2) showed patterns consistent with P lineage enrichment through the PGC stage despite no apparent enrichment in granules. 

Note: while the authors tried to maintain the orientation of embryos as depicted in the cartoons of the first slide, some embryo orientations might be flipped. 

Description of the data and file structure

Each page is an individual RNA probe, with 7 different embryo stages shown. Each image is a representative of a given stage. Brightness of the in situ may not be consistent throughout the 7 embryo stages, due to manual adjustments. Images should be used as a guide to determine where an RNA is expressed, not for intensity.

Each Group (as described in Scholl et al., 2024) is saved as a separate PDF document. Each Group is organized as in the accompanying Excel file (in situ description, same as Table S3 in Scholl et al., 2024).

Sharing/Access information

Contact corresponding author of Scholl et al., 2024 (Geraldine Seydoux, gseydoux@jhmi.edu) for requests of raw images.  MEG-3-bound RNAs were initially published in Lee at al., 2020 (https://doi.org/10.7554/eLife.52896).

Methods

For single molecule fluorescence in situ hybridization (smFISH), embryos were extruded from adult animals and subjected to freeze-crack on 0.01% poly-lysine coated slides, followed by fixation in -20°C methanol overnight. Slides were washed once in 1:1 methanol:PBSTw, five times in PBSTw (1X PBS + 0.1% Tween-20), and fixed in 4% paraformaldehyde (PFA) in PBS for 1 hour at room temperature. Fixed slides were washed four times in PBSTw, twice in 2x SSC, once in wash buffer (10% formamide, 2x SSC), and blocked in hybridization buffer (10% formamide, 2x SSC, 200ug/ml BSA, 2mM Ribonucleoside Vanadyl Complex, 0.2mg/ml yeast total RNA, 10% dextran sulfate) for 30 minutes at 37°C in a humid chamber. Slides were incubated with probes in hybridization buffer at 37°C overnight, washed twice in wash buffer at 37°C for 30 min, twice in 2x SSC, once in PBSTw, and twice in PBS before mounting in ProLong Glass Antifade Mountant with NucBlue Stain and cured overnight.

For the initial in situ screen, images were collected across 7 stages (2-cell, 4-cell, 8-cell, 28-cell, ~40-cell, ~60-cell, ~100-cell). 2-cell, 4-cell, 8-cell and 28-cell embryos were scored manually for granules visible in P1, P2, P3, and P4 blastomeres (P blastomeres are readily identified at these stages based on their position in the embryo). Transcripts positive for granules were classified as Group I or II, and transcripts negative for granules were classified as Group III. 

The distinction between Group I and Group II transcripts was made by examining ~40-cell, ~60-cell, ~100-cell stage embryos. Transcripts that showed consistent enrichment in one cell (40-cell and 60-cell stages) and two cells (100-cell stage) across these stages were classified as Group I. Because we relied on position in the embryo, rather than a marker, to identify P4 and the PGCs in these stages, we can only state that Group I transcripts exhibit patterns consistent with preferential maintenance in the P lineage to the PGC stage. Transcripts that lacked obvious enrichment at the 100-cell stage were classified as Group II (transcripts not enriched in PGCs). Five Group III transcripts (vdac-1, gld-1, F22D6.2, dlc-1, and act-2) showed patterns consistent with P lineage enrichment through the PGC stage despite no apparent enrichment in granules. 

Note: while the authors tried to maintain the orientation of embryos as depicted in the cartoons of the first slide, some embryo orientations might be flipped. 

Funding

Howard Hughes Medical Institute