DOI: 10.5061/dryad.02v6wwq98
title: Data from: Caenorhabditis elegans germ granules accumulate hundreds of low translation mRNAs with no systematic preference for germ cell fate regulators

description: 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.
description: For single molecule fluorescence <em>in situ
 </em>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 <em>in situ </em>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 P<sub>1</sub>,
 P<sub>2</sub>, P<sub>3</sub>, and
 P<sub>4</sub> 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 P<sub>4</sub> and the PGCs in these stages, we can
 only state that Group I transcripts exhibit patterns
 <em>consistent</em> 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 (<em>vdac-1, gld-1, F22D6.2,
 dlc-1, </em>and <em>act-2</em>) 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: # *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](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](mailto: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)](https://doi.org/10.7554/eLife.52896).