A novel small molecule that disrupts a key event during the oocyte-to-embryo transition in C. elegans

Abstract

The complex cellular events that occur in response to fertilization are essential for mediating the oocyte-to-embryo transition. Here, we describe a comprehensive small-molecule screen focused on identifying compounds that affect early embryonic events in Caenorhabditis elegans We identify a single novel compound that disrupts early embryogenesis with remarkable stage and species specificity. The compound, named C22, primarily impairs eggshell integrity, leading to osmotic sensitivity and embryonic lethality. The C22-induced phenotype is dependent upon the upregulation of the LET-607/CREBH transcription factor and its candidate target genes, which primarily encode factors involved in diverse aspects of protein trafficking. Together, our data suggest that in the presence of C22, one or more key components of the eggshell are inappropriately processed, leading to permeable, inviable embryos. The remarkable specificity and reversibility of this compound will facilitate further investigation into the role and regulation of protein trafficking in the early embryo, as well as serve as a tool for manipulating the life cycle for other studies such as those involving aging.

Keywords: C. elegans; Oocyte-to-embryo transition; Secretory pathway; Small molecule.

Fig. 1.

Small-molecule screen for embryonic lethality identifies C22. (A) A high-throughput screen was performed with a library of ∼37,000 small molecules, with a phenotypic readout determined by the concentration of bacterial food (represented by gray, right well) and chitinase release (represented by blue, left well) during hatching. To define positive wells, we required both low food and chitinase readings (represented by clear middle well), as an indicator that treated worms were capable of feeding but incapable of producing viable embryos. (B) Worms were treated with titrated concentrations of the 31 positive compounds identified in the primary screen (false positives not depicted). Compound treatments were visually monitored and scored for reduction of growth rate (yellow), adult/larval lethality (red), and embryonic lethality (blue) or no effect (white). The compound ChemBridge 934555, called C22, displayed only embryonic lethality at all concentrations tested (red arrowhead). (C) The structure of C22. Red arrowhead indicates functionally important methyl group identified by structure-function activity assays.

Fig. 2.

C22 rapidly and reversibly induces embryonic lethality at low doses. (A) Dosage curve measuring the live offspring produced per hour from WT hermaphrodite worms at concentrations of C22 indicated on the x-axis. Error is measured as s.d. from the mean (N=4). (B) WT L1 larvae were grown to young adult (YA) stage on plates containing either 5 μM (orange), 10 μM (green), 50 μM (yellow) C22 or a 0.2% DMSO control (blue) and then transferred to plates with 0.2% DMSO at 4 h intervals and embryonic viability was monitored. Embryonic viability is observed on all plates around 12 h post transfer and 100% viability at 16 h post transfer. Error bars represent s.d. (N=8). (C) WT L1 larvae were grown to young adult (YA) stage on plates containing 0.2% DMSO and then transferred to plates with 5 μM (orange), 10 μM (green), 50 μM (yellow) of C22 or 0.2% DMSO (blue) at 4 h intervals and embryonic viability was monitored. Embryonic lethality is observed on all plates containing C22 around 8 h post transfer and 100% C22-induced lethality is observed at 12 h post transfer. Error bars represent s.d. (N=8).

Fig. 3.

C22 exposure results in impaired eggshell integrity and CPG-2 mislocalization. (A) GFP::H2B embryos at the 2-, 4-, 8- and 12-cell stages dissected from YA worms grown in the presence of 0.2% DMSO or 5 μM C22 (N>50 per stage/condition). First row, DIC; second row, DAPI; third row, GFP::H2B; fourth row, GFP::H2B merged with DAPI images. (B) GFP::PH; mCherry::CPG-1 or GFP::PH; mCherry::CPG-2 embryos dissected from YA worms grown in the presence of 0.2% DMSO or 5 μM C22 carrying either WT let-607 or let-607(vr21). White arrow indicates peri-embryonic space in WT embryos; red arrow indicates sites where the peri-embryonic space is lost after C22 treatment. Numbers indicate embryos displaying this phenotype/number of embryos assayed. Scale bars: 10 μm.

Fig. 4.

C22-induced embryonic lethality is mediated by the CREBH transcription factor LET-607. (A) C. elegans let-607 gene structure. Annotated UTRs are labeled in gray, exons in pink and introns as lines. The vr21 mutation is located upstream, indicated by the red star. (B) Percentage of viable offspring produced from WT worms grown in the presence of 0.2% DMSO or 5 μM C22 and treated with RNAi for the empty vector L4440 (green), let-607 (blue) or a random target T01H8.2 (orange) (N≥19). (C) let-607 transcript levels relative to act-5 transcript levels (let-607/act-5) detected by RT-qPCR from WT (blue) and let-607(vr21) (purple) young adult (YA) worms, and embryos from WT (orange) and let-607(vr21) worms grown in the presence of 0.2% DMSO or 5 μM C22 (N=3 biological replicates). (D) Species-specific let-607 transcript levels relative to act-5 (let-607/act-5) detected by RT-qPCR from YA C. briggsae (green) and C. remanei (purple) worms grown in presence of 0.2% DMSO or 5 μM C22 (N=3 biological replicates). (E) Percentage of viable offspring produced from rrf-1(pk1417) worms grown in the presence of 0.2% DMSO or 5 μM C22 and treated with RNAi for the empty vector L4440 (green) or let-607 (blue) (N≥8). Error bars represent s.d. Statistical significance is indicated by star over the compared samples (**P<0.01; ***P<0.0005).

Fig. 5.

The UPR is not essential for C22-induced embryonic lethality. (A) Transcript levels for UPR markers atf-6 (purple), ire-1 (blue) and pek-1 (yellow) relative to act-5 abundance detected by RT-qPCR from young adult WT and let-607(vr21) worms grown in the presence of 0.2% DMSO or 5 μM C22. Error bars represent s.d. (N=3 biological replicates). *P<0.05. (B) PH::GFP; H2B::GFP embryos dissected from YA worms grown in the presence of either 0.2% DMSO, 5 μM C22 or 50 μg ml⁻¹ tunicamycin then stained with DAPI to determine the effect on UPR induction during eggshell formation. DIC and merged image are shown (N>50). (C) WT and let-607(vr21) worms treated with either 50 μg ml⁻¹ tunicamycin or 0.2% DMSO. Offspring viability was recorded 48 h after egg laying began. Scale bars: 10 μm (B) and 50 μm (C).

Fig. 6.

LET-607 regulates protein trafficking in C22-treated embryos. (A) Normalized sequence read abundance for LET-607::GFP ChIP-seq (green) and an input control (black) over all six C. elegans chromosomes (relative chromosome coordinates illustrated for each chromosome below). (B) Representative LET-607 binding at called targets (outlined in red), black arrow indicates direction of transcription. (C) GO analysis of called LET-607 targets for Biological Process and Cellular Component categories. Reported groups express greater than 2-fold enrichment and P<0.05 (Bonferroni corrected). (D) Transcript levels for selected LET-607 targets by RT-qPCR analysis in WT and let-607(vr21) embryos from young adult worms grown in the presence of 5 μM C22 (+) or 0.2% DMSO (−). Error bars represent s.d. (N=3 biological replicates).