Article Figures & Data
Figures
- Figure 1. A single copy system to challenge cell fate highlights robustness of differentiated cells in C. elegans.
(A) Single-cell readout cellular plasticity sensor. Cell fate–specifying transcription factors hlh-1 (MyoD homolog, inducing muscle fate) or end-1 (GATA-1 homolog, inducing intestinal fate) are induced by HS. Transcription factor ORFs are placed upstream of a trans-spliced mCherry ORF, providing a fluorescent readout. A cell fate marker (H2B::GFP) for muscle fate is integrated elsewhere in the genome. All constructs are single-copy insertions. Upon HS, red cytoplasmic fluorescence reports induction whereas green fluorescence reports muscle differentiation. (B) Muscle cell fate induction in early embryos (∼35 cell stage), DIC, and red and green fluorescence channels before and 6 h post-induction, in a control strain and upon HLH-1 ectopic expression. Upon HLH-1 expression, embryos arrest development and a number of cells express the muscle-specific marker (arrows). Scale 10 μm. (C) Brightfield images of worms ectopically expressing either mCherry or hlh-1 24 and 48 h post-induction. Bar 25 μm. (D) Number of GFP::H2B–positive cells of worms in (C). 24 h post-induction. (E) Upper left: GFP::H2B signal in an animal ectopically expressing HLH-1, 24 h post-induction (z maximal intensity projection). Bar 100 μm. Upper right: tail region; The additional green fluorescent nucleus is indicated with an arrow. Bar 10 μm. Lower left: DIC/green fluorescence overlay of the tail of an animal of the same strain, imaged 24 h post-induction of HLH-1. Bar 10 μm. Dashed line: gut, white arrow: anal sphincter cell expressing the muscle marker. Lower right: tail region, imaged 24 h post–HLH-1 induction in a strain carrying a cytoplasmic red muscle marker (myo-3p::RFP). Bar 10 μm. The cytoplasmic RFP signal outlines the characteristic saddle-like shape of the anal sphincter cell.
- Figure S1. Induced transdifferentiation during embryogenesis.
(A) GFP::H2B–positive nuclei in which ectopic expression of HLH-1 (blue) or END-1 (purple) was induced at the 8E stage of wild-type embryos, scored 24 h post-induction. (B) Phenotypic consequences of the ectopic induction of HLH-1 or mCherry in wild-type and mes-2 embryos from 2E, 4E, 8E, and 16E stage, scored 24 h post-induction. The white bars indicate larvae that develop and hatch, the gray bars indicate animals which developed but failed to hatch, and the black bars indicate animals converted to muscle lumps.
- Figure S2. Transient muscle marker expression in the anal sphincter cells.
Proportion of the population in which the anal sphincter cell expresses the muscle marker in wild-type animals after ectopic induction of HLH-1 or mCherry expression at the L1 stage, scored 12, 24, 36, and 48 h post-induction. The black bars represent the presence of myo-3p::H2B::GFP expression at the sphincter cell at a similar or stronger expression level than the weakest bona fide muscle cell in the tail region and the white bars represent an expression signal weaker than the surrounding muscle cells.
- Figure S3. Transient muscle marker expression mutants lacking H3K9 methylation or methylated H3K9 perinuclear anchoring.
Proportion of the population in which the anal sphincter cell expresses the muscle marker in set-25 met-2 or cec-4 mutant animals after ectopic induction of HLH-1 or mCherry expression at the L1 stage, scored 24 and 48 h post-induction. Scoring as in Fig S2.
- Figure 2. Absence of the Polycomb protein mes-2 renders animals sensitive to cell fate challenge.
(A) Brightfield images of mes-2 animals 24/48 h post-HS. A magnification of the gonad is shown for staging purposes (bars 25 μm). (B) Brightfield images of mes-2 animals ectopically expressing HLH-1 24/48 h post-induction. (C) Brightfield images of mes-2 animals ectopically expressing END-1 24/48 h post-induction. (D) Scoring of animal development 3 d post-induction at the first larval stage. Proportions of the populations are shown, key as indicated; L, larval stage.
- Figure 3. Cell fate challenge in the absence of Polycomb leads to hyperplasia in multiple tissues.
(A) Number of GFP::H2B–positive nuclei in wild-type and mes-2 animals upon ectopic expression of either HLH-1 or END-1 post-induction in the first larval stage. Short solid line: mean; dashed line: median. Wilcoxon test P-value 2.57 × 10−09. (B) Number of GFP::H2B–positive nuclei in different body parts of the worm, in mes-2 glo-1 control animals and animals ectopically expressing HLH-1. Wilcoxon test P-value 1.77 × 10−07 (gonad to rectum) and 4.5 × 10−06 (dorsal side). (C) Number of cells expressing markers for the E, P, M, and V lineages during development and upon HLH-1ect. induction. (D) Red fluorescence signal of the V lineage in mes-2 worms expressing HLH-1ect. or END-1ect. before and 48 h post-induction in mes-2 worms. Bar 10 μm. (E) Number of anaphases bridges in mes-2 arrested animals upon ectopic expression of HLH-1 48 h post-induction. (1, 2, 3) Magnification of anaphases bridges in animals as (C). Bar 10 μm. (F) cki-1::GFP expression and localization in mes-2 worms before and 48 h upon HLH-1ect. induction. Bar 10 μm. (G) rnr-1p::GFP expression and localization in mes-2 worms before and 48 h after HLH-1ect. induction. White arrows: seam cells, gray arrows: neurons of the ventral cord (P lineage). Bar 10 μm.
- Figure S4. Endodermal marker expression upon END-1 ectopic expression and glo-1 strain characterization.
(A) Number of cells expressing markers for the E lineage in control and upon HLH-1ect. or END-1ect. induction. Wilcoxon test P-value 0.0010. (B) Scoring of animal development 3 d post-induction at the first larval stage. Comparison of control animals and a strain ectopically expressing HLH-1 in a mes-2 and a mes-2 glo-1 genetic background. Proportions of the populations are shown. Color key as in Fig 2D. (C) GFP::H2B–positive nuclei in animals ectopically expressing HLH-1 (blue) in a mes-2 glo-1 genetic background at the first larval stage and 48 h after induction. Comparison with arrested mes-2 only L1 animals 3 d post-induction of HLH-1 expression. (D) Green fluorescence in a mes-2 animal expressing HLH-1 48 h post-induction, mes-2 glo-1 control animals post-induction, or mes-2 glo-1 48 h post-induction of HLH-1ect.. *Autofluorescence from gut granules; gonad (circled); rectum (line). Bar 10 μm.
- Figure S5. Larval arrest upon ectopic HLH-1 expression in various RNAi conditions.
Evaluation of worm development 3 d post-induction at the L1 stage of mes-2 worms after feeding parents on different RNAi conditions. Black: animals in the first larval stage; dark gray: animals in the second/third larval stage; light gray: fourth larval stage; white: adult worms.
- Figure 4. Hyperplasia is due to Notch pathway activation upon cell fate challenge.
(A) Brightfield images of rescued mes-2 mutant animals grown on lin-12(RNAi) ectopically expressing HLH-1 24/48 h post-induction. Next to the picture of the entire animal (bar 25 μm), a magnification of the gonad/vulva is shown for staging purposes (bar 25 μm). (B) Scoring of development 3 d post-induction of HLH-1 expression at the first larval stage in mes-2 animals fed on RNAi for the indicated genes. Color key in (F). (C) GFP::H2B–positive nuclei in control animals (white) and a strain ectopically expressing HLH-1 (blue) in a mes-2 genetic background fed on L4440(RNAi) and lin-12(RNAi). Comparison with arrested L1 animals 3 d post–HLH-1 induction. Wilcoxon test P-value 1.86 × 10−06 and 1.05 × 10−04 between lin-12(RNAi) rescued animals and control L4440(RNAi), and lin-12(RNAi)–arrested animals and control L4440(RNAi), respectively. (D) Quantification of the fluorescent signal from the lag-2p::GFP transgene in control (white) and upon HLH-1ect. induction (blue) in a mes-2 background before and after induction. Wilcoxon test P-values * = 0.0001, ** = 6.6 × 10−8. (E) Quantification of the fluorescent signal from the lag-2p::GFP transgene in wild-type animals. Comparison between animal control (white) and upon HLH-1ect. induction (blue). Wilcoxon test P-values ** = 6.05 × 10−10, ** = 2.35 × 10−7. The differences in lag-2p::GFP expression levels in (D) and (E) are due to image acquisition with different magnification because of animal size differences (F). Scoring of arrested animals 3 d post-induction in control strain and upon HLH-1ect. expression at the first larval stage in wild-type animals or wild-type animals with the gain-of-function lin-12(n950) mutation. Proportions of the populations are shown.
- Figure S6. Rescue of seam cells untimely division upon lin-12(RNAi) in mes-2 animals after ectopic HLH-1 expression.
(A) Red fluorescence signal of the V lineage in mes-2 lin-12(RNAi) adult worms. Comparison between a control strain and strain in which HLH-1ect. was induced at the L1 stage. (B) Number of mCherry::H2B–positive nuclei in control animals (white) and a strain in which HLH-1ect. was induced at the L1 stage (blue) in a mes-2 genetic background fed on L4440(RNAi) and lin-12(RNAi).
- Figure S7. LIN-12::GFP and lag-2p::GFP expression in mes-2 animals after ectopic HLH-1 expression.
(A) lin-12::GFP expression and localization in a mes-2 control worm before and 3 h post-HS. (B) lin-12::GFP expression and localization in a mes-2 worm before and 3 h post–muscle induction. Gonad circled with dashed line. Scale bar 10 μm. (C) lag-2p::GFP expression and localization in a mes-2 control strain and a mes-2 strain before and 48 after HLH-1ect. induction. (D) Brightfield images and lag-2p::GFP expression in wild-type animals 24 h after no HLH-1ect. induction. Arrows: somatic gonal distal tip cell. Scale bar: 25 μm. (E) Brightfield images and lag-2p::GFP expression in wild-type animals 24 h after ectopic HLH-1 induction.
- Figure 5. Starvation protects Polycomb-defective animals from cell fate challenge.
(A) Brightfield images of mes-2 mutant animals returned to feeding plates after HS in starved conditions, 24/48 h post-treatment. Magnification as in 2A (bar 25 μm). (B) Brightfield images of mes-2 mutant animals returned to feeding plates after HLH-1ect. expression in starved conditions, 24/48/72 h post-treatment. (C) Brightfield images of mes-2 mutant animals returned to feeding plates after END-1ect. expression in starved conditions, 24/48/72 h post-treatment. (D) Scoring of development 3 d post-induction at the first larval stage in starved conditions. Color key as in Fig 2D. (E) Quantification of the mCherry fluorescence from the trans-spliced ORF 6 h post-induction in fed or starved animals. (F) Scoring of animal development 3 d post-induction at the first larval stage after feeding for given times. Color key as in Fig 2D.
- Figure S8. Rescue of larval arrest and untimely seam cell division by starvation in mes-2 animals after ectopic HLH-1 expression.
(A) Brightfield images of strains expressing a green marker for muscle cells and red marker for the seam cells. Comparison between a control and a strain ectopically expressing HLH-1 in a mes-2 background 48 h post-induction in starved conditions. Scale: 25 μm. (B) Scoring of animal development 3 d post-induction of starved L1 animals. Comparison of mes-2 control animals to strains ectopically expressing HLH-1 in a mes-2 genetic background. Color key as in Fig 2D. (C) Number of nuclei expressing the muscle marker and the seam cell marker 3 d post-induction at the starved L1 stage. Comparison between a mes-2 control strain and a strain ectopically expressing HLH-1 in mes-2 background. (D) Green fluorescence signal of muscle cells and red fluorescence signal of the cells in the V lineage in mes-2 control and mes-2 animals ectopically expressing HLH-1 48 h post-induction of starved L1. *Autofluorescence from gut granules. Bar 10 μm.
- Figure S9. Larval arrest scoring in mes-2 animals after ectopic HLH-1 expression treated with various stresses.
Scoring of mes-2 animal development 3 d post-treatment at the L1 stage upon control or ectopic HLH-1 induction. Comparison between animals in which induction was performed after feeding, starvation, osmotic stress, and oxidative stress. Color key as in Fig 2D.
Supplementary Materials
Table S1 List of all candidate RNAi tested.
Table S2 Strain table.
