Insulin/IGF-dependent Wnt signaling promotes formation of germline tumors and other developmental abnormalities following early-life starvation in Caenorhabditis elegans

Abstract

The Developmental Origins of Health and Disease hypothesis postulates that early-life stressors can predispose people to disease later in life. In the roundworm Caenorhabditis elegans, prolonged early-life starvation causes germline tumors, uterine masses, and other gonad abnormalities to develop in well-fed adults. Reduction of insulin/insulin-like growth factor (IGF) signaling (IIS) during larval development suppresses these starvation-induced abnormalities. However, molecular mechanisms at play in formation and suppression of starvation-induced abnormalities are unclear. Here we describe mechanisms through which early-life starvation and reduced IIS affect starvation-induced abnormalities. Transcriptome sequencing revealed that expression of genes in the Wnt signaling pathway is upregulated in adults starved as young larvae, and that knockdown of the insulin/IGF receptor daf-2/InsR decreases their expression. Reduction of Wnt signaling through RNAi or mutation reduced starvation-induced abnormalities, and hyperactivation of Wnt signaling produced gonad abnormalities in worms that had not been starved. Genetic and reporter-gene analyses suggest that Wnt signaling acts downstream of IIS in the soma to cell-nonautonomously promote germline hyperproliferation. In summary, this work reveals that IIS-dependent transcriptional regulation of Wnt signaling promotes starvation-induced gonad abnormalities, illuminating signaling mechanisms that contribute to adult pathology following early-life starvation.

Keywords: DOHaD; L1 arrest; Wnt; diapause; early-life stress; insulin; starvation.

Fig. 1.

Expression of multiple Wnt ligands and receptors is affected by early-life starvation and IIS. a) Representative images of wild-type worms starved 1 day (control) or 8 days (starved) as L1 larvae and recovered for 90 h at 20°C with plentiful E. coli HT115 are presented. Dashed lines indicate starvation-induced abnormalities in the gonadal region, with dashes circumscribing uterine masses and germ-cell tumors. Scale bar represents 50 μm. The letter “v” indicates the vulva. b) Cartoon depicting the canonical Wnt signaling pathway is shown in the absence (left) and presence (right) of a Wnt ligand. Boxes include the genes in C. elegans that encode each component of the pathway. Adapted with permission from WormBook.org (Sawa and Korswagen 2013). c) mRNA counts per million for genes involved in the Wnt signaling pathway is plotted. The exactTest in EdgeR was used for statistics: *P < 0.05; **P < 0.01; ***P < 0.001. d) Cumulative distributions of fold changes in mRNA expression levels for starved vs control worms at egg-laying onset are plotted for genes activated by bar-1 (in red; all detected genes in black). e) Cumulative distributions of fold changes in mRNA expression levels for starved daf-2 RNAi vs starved empty vector worms at egg-laying onset is plotted for genes activated by bar-1. c–e) Samples were collected at egg-laying onset for worms starved for 1 day (control) or 8 days (starved) and recovered on empty vector or daf-2 RNAi food, as indicated in the legend (primary results are published in Jordan et al. co-submitted). d and e) The Kolmogorov–Smirnov test was used for statistics: ***P < 0.001. ev, empty vector.

Fig. 2.

Wnt signaling promotes starvation-induced gonad abnormalities. a) Frequency of all gonad abnormalities for worms starved 1 day (control) or 8 days (starved) as L1 larvae and recovered for 72 h on the indicated RNAi food is plotted. There were at least 40 worms per replicate. b) Frequency of all gonad abnormalities for control and starved worms of the indicated genotypes recovered for 72 h. There were at least 35 worms per replicate. c) Representative images of control and starved wild-type worms recovered for 90 h on bar-1 or pop-1 RNAi are presented. Images were taken at 200× total magnification. d) Representative images of control and starved gld-1(q485) mutant worms recovered for 90 h on empty vector, bar-1, or pop-1 RNAi are presented. Visible portions of the gonad are outlined with a dotted line. Images were taken at 400× total magnification. e) Frequency of large mitotic tumors for control and starved gld-1(q485) worms recovered for 90 h on empty vector, bar-1, or pop-1 RNAi are plotted. There were at least 12 worms per replicate; most replicates have >50 worms. a, b, and e) Circles represent biological replicates. Cross bars reflect the mean. *P < 0.05; **P < 0.01; ***P < 0.001; t-tests on means of replicates between empty vector RNAi and the indicated genotype or condition. c and d) Scale bars are 50 μm. The letter “v” indicates the location of the vulva. ev, empty vector.

Fig. 3.

Wnt signaling functions downstream of IIS to promote starvation-induced gonad abnormalities. a) Representative images of wild-type and heat-shock-inducible truncated bar-1 overexpression worms (conditional gain-of-function; abbreviated “bar-1 OE”) exposed to the indicated conditions and recovered for 90 h. Images taken at 200× total magnification. b) Frequency of all gonad abnormalities in control and starved worms recovered for 90 h in the indicated conditions. There were at least 37 worms per replicate. c) Representative images of gld-1(q485) mutant and gld-1(q485); bar-1 overexpression worms exposed to the indicated conditions and recovered for 90 h. Images taken at 400× total magnification. d) Frequency of large mitotic tumors for control and starved worms recovered for 90 h in the indicated conditions is plotted. There were at least 36 worms per replicate. e) Frequency of all gonad abnormalities for control and starved wild-type and cwn-1(ok546) mutant worms recovered for 90 h in the indicated conditions is plotted. There were at least 45 worms per replicate. f) Frequency of large mitotic tumors for control and starved gld-1(q485) and gld-1(q485); cwn-1(ok546) worms recovered for 90 h in the indicated conditions is plotted. There were at least 30 worms per replicate. g) Frequency of all gonad abnormalities for starved wild-type and cwn-1(ok546) mutant worms recovered for 72 h in the indicated conditions is plotted. There were at least 28 worms with an average of 45 per replicate. a and c) Scale bars represent 50 μm. The letter “v” indicates the location of the vulva. b, d, e, f, and g) Circles represent biological replicates. Cross bars indicate the mean. *P < 0.05; **P < 0.01; ***P < 0.001; t-test on means of replicates. ev, empty vector; ns, not significant.

Fig. 4.

Early-life starvation and insulin/IGF signaling promote cwn-1 reporter-gene expression. Background-corrected mean pixel intensity is plotted for CWN-1::VENUS in control and starved worms recovered for 48 h in the conditions indicated. There is a single data point outside the plotted range for control empty vector and starved daf-2. Each point represents an individual worm. Cross bars indicate the mean. Four biological replicates were performed and pooled for display. ***P < 0.001; pairwise linear mixed-effect model. ev, empty vector.

Fig. 5.

cwn-1 and cfz-2 function in somatic tissues to promote starvation-induced gonad abnormalities. Frequency of all gonad abnormalities for control and starved wild-type, rrf-1(pk1417), and ppw-1(pk2505) mutant worms recovered for 90 h in the indicated conditions is plotted. There were at least 50 worms per replicate. Circles represent biological replicates. Cross bars indicate the mean. **P < 0.01; ***P < 0.001; t-test on means of replicates. ev, empty vector; ns, not significant.