Article Figures & Data
Figures
- Figure 1. Hypoxia represses RAS/MAPK–mediated differentiation in different tissues.
(A) Overview of vulval development showing the known interactions between the RAS/MAPK and DELTA/NOTCH pathways. During the L2 stage, LIN-3 EGF activates the RAS/MAPK cascade in P5.p-P7.p. P6.p, in which RAS/MAPK activity is highest, adopts the 1° cell fate, and expresses DSL NOTCH ligands to activate LIN-12 NOTCH signaling in the adjacent Pn.p cells, which adopt the 2° fate. The uninduced P(3-4).p and P8.p cells adopt the 3° fate and fuse with the surrounding hypodermis. (B) Vulval phenotypes of the let-60 ras G13E mutation in the N2 Bristol (left) and CB4856 Hawaii (right) background with varying oxygen concentrations. Solid lines indicate induced 1° and 2° and arrowheads uninduced 3° VPCs in L4 larvae. (C) VI of N2 Bristol and CB4856 Hawaii let-60 ras G13E mutants raised in varying oxygen concentrations. (D) Effect of hypoxia on different RTK/RAS/MAPK pathway, bar-1(ga80), lin-12(n137), and lin-12(n137n720) mutants. ∆VI indicates the change in VI of animals raised in 0.5% compared with controls grown in 21% oxygen. ∆%Muv and ∆%Vul indicate the change in the percentage of animals with VI > 3 and VI < 3, respectively. The absolute VIs at 0.5% oxygen are shown in the rightmost red column. (E) Suppression of the stacked oocyte phenotype in let-60(ga89gf) animals raised at the restrictive temperature by hypoxia. Arrowheads point at the stacked oocytes formed in the proximal gonad under normoxia. (F) Suppression of the duct cell duplication phenotype in let-60(n1046gf) mutants by hypoxia. Arrows point at the duct cell nuclei expressing LIN-48::GFP formed under normoxia (top) and hypoxia (bottom). (C, D) Error bars in (C) and (D) indicate the 95% confidence intervals, and P-values, indicated with ***P < 0.001 and **P < 0.01, were derived by bootstrapping 1,000 samples. (E, F) In (E) and (F), error bars indicate the standard error of the mean, and P-values were calculated with a Fisher’s exact test. The numbers of animals scored are indicated in brackets. The scale bars represent 5 μm.
- Figure 2. The hypoxia-response pathway negatively regulates VI under normoxia.
(A) Schematic overview of the conserved hypoxia-response pathway. The C. elegans gene names are indicated. (B) Vulval phenotypes of double and triple mutants between let-60(n1046gf) and components of the hypoxia-response pathway under normoxia. Solid lines indicate induced 1° and 2° and arrowheads uninduced 3° VPCs in the L4 larvae. (C) Mutations in the hypoxia-response pathway change the VI of let-60(n1046gf) mutants. ∆VI indicates the change in VI of the genotypes relative to let-60(n1046gf) single mutant siblings obtained from the crosses. ∆%Muv indicates the change in the percentage of animals with VI > 3. The absolute VIs of the double/triple mutants are shown in the rightmost blue column. (D) Overexpression of wild-type egl-9::gfp increases the VI. ∆VI indicates the change in VI of animals carrying a wild-type (dark bars) or hydroxylase deficient (light bars) multi-copy egl-9::gfp array compared with siblings without array. Error bars indicate the 95% confidence intervals. P-values, indicated with ***P < 0.001 and **P < 0.01, were derived by bootstrapping 1,000 samples. (E–I, J) MPK-1 biosensor (ERK-nKTR) activity values measured in the VPCs of mid-L2 larvae with the indicated mutant backgrounds and (J) comparison of the MPK-1 activity levels in P6.p across the different genotypes. Relative MPK-1 activity values were measured as described under the Materials and Methods section. The numbers of animals scored are indicated in brackets. The scale bar represents 5 μm.
- Figure S1. Comparison of the zh111 and ia04 deletions in the hif-1 locus.
(A) Schematic representation of the hif-1 locus with the five isoforms shown in grey. The canonical deletion allele ia04 is depicted in light blue and the larger zh111 deletion is shown in dark blue. Note that the zh111 deletion affects all isoforms as opposed to ia04. (B) The zh111 allele has a stronger effect on the VI in the let-60(n1046gf) background than ia04. (B) Error bars in (B) indicate the 95% confidence intervals, and P-values, indicated with ***P < 0.001 and **P < 0.01, were derived by bootstrapping 1,000 samples.
- Figure S2. Genetic interaction between egl-9(sa307lf) and RAS/MAPK pathway mutations.
∆VI indicates the change in VI of the indicated genotypes in the egl-9(sa307lf) background compared with single (or double) mutant siblings obtained from the crosses. ∆%Muv and ∆%Vul indicate the changes in the percentage of animals with VI > 3 and VI < 3, respectively. The absolute VIs of the double (or triple) mutants are shown in the rightmost column. Error bars indicate the 95% confidence intervals. P-values, indicated with **P < 0.01, were derived by bootstrapping 1,000 samples. The numbers of animals scored are indicated in brackets.
- Figure 3. egl-9 is a NOTCH target that promotes HIF-1 degradation in the induced VPCs.
(A) HIF-1::GFP expression in wild-type, egl-9(sa307lf), and vhl-1(ok161lf) larvae before VI. Arrowheads point at the nuclei of the VPCs. (B) Pn.p cell– and gut-specific hif-1 RNAi (see main text and the Material and Methods section). ∆VI indicates the change in VI in hif-1 RNAi compared with empty vector–treated control animals. Error bars indicate the 95% confidence interval, and P-values were derived by bootstrapping 1,000 samples. (C) EGL-9::GFP expression in a wild-type mid-L2 larva (top panel) at the onset of VI, a late-L2/early-L3 larva before the proximal and after the distal VPCs had divided (second panel) and a mid-L3 larva after all VPCs had divided once (third panel, Pn.px stage). The arrowheads point at the VPC nuclei and the small arrows at the nuclei of ventral nerve cord neurons expressing EGL-9::GFP. Expression in let-60 ras(n2021rf), lin-12 notch(n137n720lf), and lin-12 notch(n137gf) mutants is shown in mid-L3 larvae, after the first round of VPC divisions had been completed (Pn.px stage). Induced VPCs are underlined. The scale bar represents 5 μm. (D) Schematic representation of the wild-type EGL-9::GFP pattern during vulval development. (E–G) Quantification of EGL-9::GFP expression in the four genotypes shown in (C) after VI (early to mid L3 and at the Pn.pxx stage in (G)). Error bars indicate the standard error of the mean, and P-values, indicated with ***P < 0.001 and *P < 0.05, were calculated in a two-tailed t test. The numbers of animals scored are indicated in brackets.
- Figure S3. Enrichment of predicted LAG-1–binding sites in the egl-9 locus.
(A) Predicted CSL-binding sites defined by the RTGGGAA motif in the egl-9 genomic locus. (B) The location of each CSL site is indicated relative to the transcriptional start site and the consensus sequence is present either in the direction of transcription (“+”) or against (“−”).
- Figure S4. EGL-9 does not modulate LIN-12 NOTCH signaling.
The absolute VIs of lin-12(n137gf) and lin-12(n137n720lf) mutants in the egl-9(sa307lf) background compared with egl-9(+) single mutant siblings obtained from the crosses are shown. %Muv and %Vul indicate the percentage of animals with VI > 3 and VI < 3, respectively. Error bars indicate the 95% confidence intervals. P-values were derived by bootstrapping 1,000 samples. The numbers of animals scored are indicated in brackets.
- Figure 4. The HIF-1 target NHR-57 inhibits RAS/MAPK signaling under normoxia and hypoxia.
(A) Knock-down of nhr-57 increases the VI of let-60(n1046gf) in an egl-9(sa307lf) but not in a hif-1(zh111lf) background. ∆VI and ∆%Muv indicate the changes in VI and percentage of animals with VI > 3 after nhr-57 RNAi compared with empty vector–treated control animals. (B) Deletion of the nhr-57(tm4533lf) allele increases the VI of let-60(n1046gf) single and let-60(n1046gf); egl-9(sa307lf) double mutants but has no effect in a let-60(n1046gf); hif-1(zh111lf) background. ∆VI and ∆%Muv indicate the changes in VI and the percentage of animals with VI > 3 compared with nhr-57(+) control siblings. (C) Hypoxic treatment decreases the VI in the hif-1(ia04lf), hif-1(zh111lf), and egl-9(sa307lf) hif-1(ia04lf) but not the nhr-57(tm4533lf) background. ∆VI and ∆%Muv indicate the changes in VI and percentage of animals with VI > 3 raised in hypoxia compared with normoxia. Error bars indicate 95% confidence intervals, and P-values, indicated with ***P < 0.001 and **P < 0.01, were derived by bootstrapping 1,000 samples. The numbers of animals scored are indicated in brackets. (D) NHR-57::GFP expression pattern in the wild-type (left panels) and egl-9(sa307lf) (right panels) background in late-L2/early-L3 larvae at the Pn.p stage (top panels), in mid L3 larvae (Pn.px stage) after induction (middle panels), and at the end of vulval differentiation (bottom panels). Arrowheads and solid lines point at the nuclei of the induced or uninduced VPCs and their descendants, respectively. The scale bar represents 5 μm. (E) Schematic representation of the NHR-57::GFP pattern during vulval development in the wild-type (left) and in an egl-9(sa307lf) background (right).
- Figure 5. EGL-9 inhibits the HIF-1 target NHR-57 from reducing RAS/MAPK signaling through a DELTA/NOTCH-induced negative feedback loop.
(A) Model illustrating the cross-talk of the hypoxia-response, DELTA/NOTCH, and RAS/MAPK pathways at normoxia. At the onset of VI, before a distinction between 1° and 2° VPCs is made, lateral DELTA/NOTCH signaling between the proximal VPCs induces EGL-9 expression to maintain low HIF-1 and NHR-57 levels, thereby keeping the VPCs competent to respond to RAS/MAPK signaling. RAS/MAPK signaling, in turn, promotes the expression of the DELTA family NOTCH ligands. Distal VPCs lose their competence because of higher NHR-57 levels and adopt the 3° fate. Thereafter, NOTCH signaling directly induces the 2° fate and inhibits RAS/MAPK signaling in P5.p and P7.p. (B) Under hypoxia, the inhibition of HIF-1 and NHR-57 by EGL-9 is reduced because of the lack of oxygen and vulval fate acquisition is compromised. An as-of-yet unidentified factor activates NHR-57 during hypoxia independently of HIF-1.
Supplementary Materials
Table S3 List of strains used.
Table S4 Complete dataset of VI counts.
Supplemental Data 1.
Supplemental Data 2.
