Specific sensory neurons and insulin-like peptides modulate food type-dependent oogenesis and fertilization in Caenorhabditis elegans
- Department of Biological Sciences, Wayne State University, United States
- Centre for Developmental Neurobiology, King’s College London, United Kingdom
Figures
Figure 1
C. elegans exhibit early oogenesis and faster fertilization rate in response to a specific bacterial food source.
(A) The C. elegans reproductive developmental program, which depicts the timing of the spermatogenesis-to-oogenesis switch at L4. (B) Somatic development was unchanged between animals fed the two Escherichia coli food types (n=286 on OP50; n=265 on CS180; p=0.72). (C–E) Worms fed OP50 had more progeny (C), slower fertilization rates (D), and more sperm (E) than worms fed CS180. (F) Simplified illustration of vulval morphology at various substages of L4, where the nuclei of specific cells are shown (adapted from Mok et al., 2015). Red arrowheads indicate the finger-like structures at the sides of the vulva, which migrate ventrally during late L4. Lethargus is the molting period from L4 to young adulthood. (G) Early onset of oogenesis was determined by the initial expression of lin-41::GFP. Anterior is to the left and dorsal is to the top of each panel. Scale bar is 10 μm. (H–I) Worms on CS180 began oogenesis earlier at mid-L4 than worms on OP50, based on the earliest lin-41::GFP (H) or oma-1::GFP (I) expression. Early oogenesis onset at mid-L4 is highlighted by a dotted box in these panels and later panels. (J) Adults on CS180 had more arrested oocytes than age-matched adults on OP50. Statistical analyses for (C, D, H and I) are in Figure 1—source data 1. For (E), n of animals on OP50 is 14; n on CS180, 12. For (J), n of animals on OP50 is 23; n on CS180, 31. Error bars in (C–D) represent standard deviation; (E, J) SEM. ** indicates p<0.01 and ***, p<0.001.
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Figure 1—source data 1
Statistical analyses of wild-type progeny, fertilization rates, and oogenesis onset on OP50 versus CS180.
Sheet 1 shows the analyses for total progeny; sheet 2, for fertilization rates; and sheet 3, for onset of oogenesis. On sheets 1 and 2, N is the total number of animals assayed. Means are from pooled data from multiple, independent trials. SD is the standard deviation. On sheet 3, GFP (+) and GFP (-) are animals that expressed or did not express the indicated oogenesis markers, respectively. On all sheets, significant p values are shown in red and italicized. Figure 1 panels associated with the data are indicated. WT denotes wild type. * denotes p values between OP50-fed worms versus CS180-fed worms.
- https://cdn.elifesciences.org/articles/83224/elife-83224-fig1-data1-v2.xlsx
Figure 2
The CS180 effects on oogenesis are independent of lipopolysaccharide (LPS) length and absent in HT115 and other bacteria.
(A) The LPS of E. coli B and K-12 strains. CS2429 is derived from CS180 and has a truncated LPS (Maier et al., 2010, and references therein). The dotted blue line indicates the site of truncation. (B–D) The shorter LPS of CS2429 elicited the same total progeny (B), a similar pattern of oogenesis reporter expression (C), and the same rate of fertilization (D), as the longer LPS of the parent strain CS180. (E–F) C. elegans oogenesis onset (E) and fertilization rates (F) on the E. coli K-12 HT115 bacteria were compared to those of C. elegans on OP50 and CS180. (G–H) Oogenesis at mid-L4 (G) and fertilization rates at 22 hr (H) on OP50 and CS180 were compared to oogenesis onset (G) and fertilization rates (H) on OP50 that had been supplemented with: (i) CS180-conditioned Luria broth (LB) media (med) or (ii) CS180-conditioned media that was filtered through a 0.45 μm nylon membrane (filtrate). For comparison, worms on OP50 that had been supplemented with LB alone or with LB that was filtered through a nylon membrane are also shown. (I) Two strains of the pathogen S. marcescens, Db1140 and Db11, did not induce early oogenesis. See Figure 2—source data 1 for the sample sizes and complete statistical analyses of the data in this figure. Error bars represent standard deviations. * indicates p<0.05; **, p<0.01; ***, p<0.001; and suppl, supplementation.
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Figure 2—source data 1
Statistical analyses of wild-type progeny, oogenesis onset, and fertilization rates on OP50, lipopolysaccharides (LPS) mutant bacteria, HT115, S. marcescens, and CS180-conditioned media.
Sheet 1 shows the analyses for total progeny; sheet 2, for onset of oogenesis; and sheet 3, for fertilization rates. On sheets 1 and 3, N is the total number of animals that were assayed. Means are from pooled data from multiple, independent trials. SD is the standard deviation. On sheet 2, GFP (+) and GFP (-) are animals that expressed or did not express the lin-41::GFP oogenesis marker, respectively. On all sheets, significant p values are shown in red and italicized. Figure 2 panels associated with the data are indicated. WT denotes wild type. NA means not applicable.
- https://cdn.elifesciences.org/articles/83224/elife-83224-fig2-data1-v2.xlsx
Figure 3
Food type modulates oogenesis onset during L3 and fertilization rates after early L4.
Wild-type C. elegans were shifted between E. coli OP50 and CS180 at different stages of larval development. (A–B) The critical period for food-type modulation of total progeny (A) and oogenesis onset (B) is around late L3. (C–F) The critical period for modulation of fertilization is after early L4. See Figure 3—source data 1 for the sample sizes and complete statistical analyses of the data in this figure. Error bars represent standard deviations. * indicates p<0.05; **, p<0.01; ***, p<0.001; ns, not significant; and leth, lethargus.
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Figure 3—source data 1
Statistical analyses of the progeny, oogenesis onset, and fertilization rates in food-switched versus non-switched wild-type animals.
Sheet 1 shows the analyses for total progeny; sheet 2, for onset of oogenesis; sheet 3, for fertilization rates before normalization; and sheet 4, for normalized fertilization rates. On sheets 1 and 3, N is the total number of animals that were assayed. Means are from pooled data from multiple, independent trials. SD is the standard deviation. On sheet 2, GFP (+) and GFP (-) are animals that expressed or did not express the lin-41::GFP oogenesis marker, respectively. On all sheets, significant p values are shown in red and italicized. Figure 3 panels associated with the data are indicated. Symbols, abbreviations, and superscripted letters are described within each sheet.
- https://cdn.elifesciences.org/articles/83224/elife-83224-fig3-data1-v2.xlsx
Figure 4
The ASJ sensory neuron and insulin-like peptide (ILP) INS-6 alter total progeny in response to food type.
(A) Food-type differences in total progeny were lost in sensory mutants osm-3 and odr-1 and in animals lacking the ASJ neurons. (B) Total progeny of the strong reduction-of-function mutant daf-2(e1370) and the daf-16(mu86) loss-of-function mutant on OP50 and CS180. (C–D) OP50- and CS180-dependent progeny of ILP deletion mutants, daf-28(tm2308) and ins-1(nr2091) (C) and ins-6(tm2416) (D). (E) Epistasis analysis between the food type-dependent total progeny phenotypes of daf-16(mu86) and ins-6(tm2416). While the animals in (A) and (C–E) were grown continuously at 25°C, the animals in (B) were shifted from 20°C to 25°C at the early L3 stage, because daf-2 mutants arrest developmentally prior to the L3 stage at 25°C but not at 20°C. See Figure 4—source data 1 for the sample sizes and complete statistical analyses of the data in this figure. Error bars represent standard deviations. ** indicates p<0.01; ***, p<0.001; and ns, not significant.
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Figure 4—source data 1
Statistical analyses of total progeny of different groups of animals on OP50 versus CS180.
N is the total number of mothers that were assayed. Means are from pooled data from multiple, independent trials. SD is the standard deviation. Significant p values are shown in red and italicized. Figure 4 panels associated with the data are indicated. * denotes p values between OP50-fed worms versus CS180-fed worms. # denotes experiments in which animals that carry the daf-2(e1368), daf-2(e1370), or daf-16(mu86) mutation and the corresponding wild-type controls were grown at 20°C and transferred to 25°C at the early L3 stage. All other experiments involved animals that were continuously grown at 25°C.
- https://cdn.elifesciences.org/articles/83224/elife-83224-fig4-data1-v2.xlsx
Figure 5
ASJ neurons and ins-6 promote early oogenesis onset.
(A) Food type-dependent expression of the oogenesis reporter lin-41::GFP in control and different sensory mutants. (B–D) lin-41::GFP expression in daf-2 reduction-of-function mutants (B), daf-16(mu86) (C), and in insulin-like peptide (ILP) deletion (Δ) mutants (D). (E) Food type-dependent expression of a second oogenesis reporter, oma-1::GFP, in control, ASJ-ablated and ins-6(tm2416) worms. (F) Loss of daf-16 did not rescue the delay in oogenesis onset in ins-6(tm2416) mutants. Controls and daf-2 mutants in (B) were shifted from 20°C to 25°C as early L3s. In contrast, all other animals were continuously grown at 25°C on OP50 (white bars) or CS180 (gray bars). See Figure 5—source data 1 for the sample sizes and complete statistical analyses of the data in this figure. * indicates p<0.05; **, p<0.01; ***, p<0.001; and leth, lethargus.
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Figure 5—source data 1
Statistical analyses of oogenesis onset in different groups of animals on OP50 versus CS180.
GFP (+) and GFP (-) are animals that expressed or did not express the oogenesis markers, lin-41::GFP or oma-1::GFP, respectively. Figure 5 panels associated with the data are indicated. Significant p values are shown in red and italicized. * denotes p values between OP50-fed worms versus CS180-fed worms. # denotes experiments in which animals that carry the daf-2(e1368) or daf-2(e1370) mutation and the corresponding controls were grown at 20°C and transferred to 25°C at the early L3 stage. All other experiments involved animals that were continuously grown at 25°C.
- https://cdn.elifesciences.org/articles/83224/elife-83224-fig5-data1-v2.xlsx
Figure 6
ins-6 acts from ASJ neurons to promote early oogenesis.
(A) ins-6p::mCherry transcriptional reporter drcSi68 expression in ASI (n=29, OP50; n=29, CS180) and ASJ (n=30, OP50; n=30, CS180) neurons during mid-to-late L3. (B) lin-41::GFP expression in ins-6(tm2416) deletion mutants that were either rescued with: (i) the wild-type ins-6 genomic locus in two independent lines, jxEx27 and jxEx28; (ii) ASI neuron-specific expression of ins-6; or (iii) ASJ neuron-specific expression of ins-6 in two independent lines, jxEx58 and jxEx59. (C) lin-41::GFP expression in the (i) loxP-flanked ins-6(syb7547) homozygotes, (ii) ins-6(tm2416) homozygotes that carry the CRE recombinase transgene that is specifically expressed in ASJ (ASJp::CRE), (iii) ins-6(tm2416) heterozygotes that carry the ASJp::CRE transgene, (iv) ins-6(tm2416)/ins-6(syb7547) transheterozygotes, and (v) animals in which ins-6 was deleted specifically from ASJ but remains present in ASI. See Figure 6—source data 1 for the sample sizes and complete statistical analyses of the data in this figure. * indicates p<0.05; **, p<0.01; ***, p<0.001; and leth, lethargus.
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Figure 6—source data 1
Statistical analyses of oogenesis onset in different groups of animals on OP50 versus CS180.
GFP (+) and GFP (-) are animals that expressed or did not express the oogenesis marker lin-41::GFP, respectively. Figure 6 panels associated with the data are indicated. Significant p values are shown in red and italicized. * denotes p values between OP50-fed worms versus CS180-fed worms.
- https://cdn.elifesciences.org/articles/83224/elife-83224-fig6-data1-v2.xlsx
Figure 7
AWA olfactory neurons modulate food type-dependent fertilization rates, but not early oogenesis onset.
(A–B) Sensory mutations influenced the food-type effects on fertilization rates. (C–I) Animals that lack the sensory neurons ADF (C), ASH (D), ASI (E), ASK (F), ASJ (G), AWB (H), and AWC (I). (J) Animals lacking the sensory neuron AWA in odr-7 mutants. (K) Animals that lack both AWA and ASJ. Controls for (G and K) carried the ofm-1p::gfp marker in the wild-type background. All other controls (A–F, H–J) were wild-type worms. (L) Food type-dependent expression of the oogenesis reporter lin-41::GFP in controls or animals lacking AWA and/or ASJ. See Figure 7—source data 1 for the sample sizes and complete statistical analyses of the data in this figure. ** indicates p<0.01; ***, p<0.001; and leth, lethargus.
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Figure 7—source data 1
Statistical analyses of fertilization rates and oogenesis onset in different sensory-impaired animals on OP50 versus CS180.
Sheet 1 shows the analyses for fertilization rates before normalization; sheet 2, for normalized fertilization rates; and sheet 3, for oogenesis onset in animals lacking AWA and/or ASJ. On sheet 1, N is the total number of animals that were assayed. Means are from pooled data from multiple, independent trials. SD is the standard deviation. On sheet 3, GFP (+) and GFP (-) are animals that expressed or did not express the lin-41::GFP oogenesis marker, respectively. On all sheets, Figure 7 panels associated with the data are indicated. Significant p values are shown in red and italicized. On sheets 1 and/or 3, the following abbreviation and superscripted letters denote: WT, wild type; NA, not applicable; A, p values between a given strain grown on OP50 versus CS180 at each time point; B, p values between a given strain grown on OP50 versus wild type on OP50; and C, p values between a given strain grown on CS180 versus wild type on CS180.
- https://cdn.elifesciences.org/articles/83224/elife-83224-fig7-data1-v2.xlsx
Figure 8
Insulin receptor signaling alters global fertilization rates independent of food type.
(A) The weak reduction-of-function daf-2(e1368) mutant. (B) The strong reduction-of-function daf-2(e1370) mutant. (C–F) daf-16 null mutants and the three insulin-like peptide (ILP) mutants, ins-6, daf-28, or ins-1. Controls and daf-2 mutants in (A–B) were shifted from 20°C to 25°C as early L4s, whereas all other animals were continuously grown at 25°C on OP50 (black closed circles for wild type; black closed triangles for the indicated mutants) or CS180 (gray open circles for wild type; blue open triangles for the indicated mutants). See Figure 8—source data 1 for the sample sizes and complete statistical analyses of the data in this figure.
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Figure 8—source data 1
Statistical analyses of fertilization rates in insulin signaling-impaired animals on OP50 versus CS180.
Sheet 1 shows the analyses for fertilization rates before normalization; and sheet 2, for normalized fertilization rates. N is the total number of animals that were assayed. Means are from pooled data from multiple, independent trials. SD is the standard deviation. Figure 8 panels associated with the data are indicated. Significant p values are shown in red and italicized. # denotes experiments in which animals that carry the daf-2(e1368) or daf-2(e1370) mutation and the corresponding controls were grown at 20°C and transferred to 25°C at the early L4 stage. The following abbreviation and superscripted letters denote: WT, wild type; NA, not applicable; A, p values between a given strain on OP50 versus CS180 at each time point; B, p values between a given strain grown on OP50 versus wild type on OP50; and C, p values between a given strain grown on CS180 versus wild type on CS180; D, p values between a given strain versus the indicated insulin-like peptide (ILP) double or triple mutant on OP50; and E, p values between a given strain versus the indicated ILP double or triple mutant on CS180.
- https://cdn.elifesciences.org/articles/83224/elife-83224-fig8-data1-v2.xlsx
Figure 9
A model for the sensory modulation of food type-dependent oogenesis onset and fertilization.
Insulin-like peptide (ILP) INS-6 acts from ASJ neurons to promote early onset of oogenesis. Olfactory neuron AWA promotes oocyte fertilization through mechanism(s) distinct from DAF-2 insulin receptor signaling, which in turn regulates fertilization independent of food type through the activities of other ILP(s).
Additional files
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MDAR checklist
- https://cdn.elifesciences.org/articles/83224/elife-83224-mdarchecklist1-v2.pdf
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Supplementary file 1
List of strains used in the study.
- https://cdn.elifesciences.org/articles/83224/elife-83224-supp1-v2.docx
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Specific sensory neurons and insulin-like peptides modulate food type-dependent oogenesis and fertilization in Caenorhabditis elegans
eLife 12:e83224.
https://doi.org/10.7554/eLife.83224
