The nuclear accumulation of N-MYRF-1 coincides with the upregulation of lin-4 in developmental timing.

A. Nuclear localization of GFP::MYRF-1 is increased in late L1, coinciding with the upregulation of Plin-4-GFP. GFP, endogenously inserted at MYRF-1 Ala171, labels both full-length MYRF-1 and post-cleaved N-MYRF-1. Plin-4-GFP(maIs134) is a transcriptional reporter of lin-4, carrying a 2.4 kb sequence upstream of the lin-4 gene that drives GFP. While GFP::MYRF-1 is initially localized at the cell membrane in early-mid L1 (6 post-hatch hours), it becomes enriched in the nucleus towards late L1 (15 post-hatch hours). Plin-4-GFP is barely detected in early L1 but is upregulated in late L1. The insert shows a zoomed-in view of the framed area, covering part of the pharynx. B. Quantification of animals showing a particular pattern of GFP::MYRF-1 (as shown in (A)) at various stages. The number of animals analyzed is indicated on each bar. C. The fluorescence intensity of the lin-4 transcriptional reporter (as shown in (A)) was quantified and presented as mean ± SEM (t-test, ****p < 0.0001); n > 20, where n is the number of nematodes analyzed.

MYRF is required for lin-4 upregulation during late L1

A. Plin-4-GFP is not upregulated in myrf-1(ju1121). The expression of the lin-4 transcriptional reporter in wild-type and myrf-1(ju1121) animals was examined at the early L1 (0h), late L1 (16h), and early L2 stages (21h). B. The fluorescence intensity of the lin-4 transcriptional reporter (as shown in (A)) was quantified and presented as mean ± SEM (t-test, ****p < 0.0001). More than 20 animals were analyzed. C. The endogenous lin-4 transcriptional reporter lin-4p::nls::mScarlet(umn84) is not upregulated in myrf-1(ju1121) except for a few pharyngeal nuclei. The expression of the lin-4p::nls::mScarlet in wild-type and myrf-1(ju1121) animals was examined at the early-mid L1 (6h), late L1 (14h), and early L2 stages (21h). D. The fluorescence intensity of lin-4p::nls::mScarlet(umn84) (as shown in (C)) was quantified and presented as mean ± SEM (t-test, ****p < 0.0001). The “whole body” refers to the region of interest (ROI), which encompasses the entire body of the animal. The ROI of “in head neurons” comprises multiple head neuron nuclei found in each animal. For “in pharyngeal cells”, the ROI includes the 8-9 pharyngeal nuclei that exhibit bright mScarlet signals. More than 15 animals were analyzed. E. The abundance of mature lin-4 miRNAs in wild-type and myrf-1(ju1121) animals was examined at the early L1 (0h), late L1 (16h), and early L2 stages (21h) using qPCR analysis with probes specifically detecting lin-4 microRNA. Each data point represents relative fold change of each sample comparing to the wild type Early L1 sample within one set of experiment. The data represent three biological replicates. Statistics used t-test. *p < 0.05, ***p < 0.001.

There is a sustained high level of LIN-14 protein in myrf-1 mutants.

A. Expression of LIN-14::GFP(cc2841) in wild-type and myrf-1(ju1121) at the early L1 (0h), late L1 (16h), and early L2 (21h) stages. GFP was endogenously tagged at the LIN-14 C-terminus. LIN-14::GFP is bright in early L1 and downregulated in late L1. LIN-14::GFP is not affected by myrf-1(ju1121) at early L1 but significantly brighter than wild-type control at late L1 and L2. B: The fluorescence intensity for LIN-14::GFP (as shown in (G)) was measured and presented as mean ± SEM (t-test, ns: not significant, p > 0.05; ***: p < 0.001) from the analysis of more than 20 nematodes. Each data point represents the mean intensity of the head region in an individual animal. The head region was selected due to its low autofluorescence background

MYRF-1 is sufficient to drive lin-4 expression in a cell-autonomous manner.

A. Genetic rescue of MYRF-1 in myrf-1(ju1121) using tissue-specific promoters. Plin-4-GFP(maIs134) signals are observed specifically in body wall muscles and epidermis (asterisk) of myrf-1(ju1121) carrying transgene Pmyo-3-myrf-1 and Pdpy-7-myrf-1, respectively, while no detectable Plin-4-GFP is observed in L2 of myrf-1(ju1121). B. Tissue-specific ablation of myrf-1 in the epidermis. myrf-1LoxP(ybq98) combined with Pdpy-7-NLS::Cre(tmIs1028) caused loss or drastic decrease of Plin-4-GFP(maIs134) in the epidermis, while signals were detected in other tissues. Representative images of L2 (24h) animals are shown. C. Fluorescence intensity measurements (as shown in (B)) are displayed for a ROI bar drawn transversely and centered at one seam cell. Individual lines represent signals from one animal.

Hyperactive MYRF-1 drives premature expression of lin-4.

A. The reporter of lin-4 transcription, labeled by endogenously inserted nls::mScarlet (umn84), which also produces a loss-of-function allele of lin-4. The fluorescence was not observed in embryos or early L1, but in late L1, confirming the previous reports. B. Overexpression of a hyperactive MYRF-1 mutant, GFP::MYRF-1(delete 601-650) caused premature lin-4 transcription in embryos and early L1, labeled by lin-4p::nls::mScarlet. C. The expression of Plin-4-GFP(maIs134) in wild type and myrf-1(syb1313) mutants. At 6 hours, Plin-4-GFP expression is elevated in the neurons of myrf-1(syb1313) mutants but undetectable in wild type. By late L1 (15h), Plin-4-GFP is upregulated in multiple tissues in wild type. Although GFP expression is sustained in neurons (arrow) of the mutants, it is significantly weak or absent in the pharynx (asterisk) of the mutants. D. The fluorescence intensity of the lin-4 transcriptional reporter (as displayed in (C)) was measured and presented as mean ± SEM (t-test, *p < 0.05, **p < 0.01, ***p < 0.001). Each data point represents the mean intensity of the head neurons or pharynx region in individual animal, which were imaged using confocal microscopy. E. The expression of lin-4p::nls::mScarlet(umn84) in wild type and myrf-1(syb1313) mutants. At 6 hours, mScarlet expression is elevated in certain neurons of myrf-1(syb1313) mutants but undetectable in wild type. By late L1 (14h), mScarlet is upregulated in multiple tissues in both wild type and myrf-1(syb1313) mutants. The mutants exhibit stronger mScarlet signals than wild type. F. The fluorescence intensity of lin-4p::nls::mScarlet (as displayed in (E)) was measured and presented as mean ± SEM (t-test, *p < 0.05, **p < 0.01, ***p < 0.001). The ROI for “whole body” encompasses the entire body of the animal. The ROI of “in head neurons” comprises multiple head neuron nuclei in each animal. The ROI for “in pharyngeal cells” includes the 8-9 pharyngeal nuclei that exhibit strong mScarlet signals. Each data point represents the mean intensity of the ROI in individual animal.

lin-4 promoter DNA recruits MYRF-1 protein in vivo.

A. A subset of animals carrying the maIs134 transgene constitutive dauer formation even in the absence of food depletion on culture plates. Dauer formation was assessed by treating the animals with 1% SDS for 20 minutes. MYRF-1 overexpression (ybqIs112) suppresses the constitutive dauer formation in maIs134. Animals were cultured at 20°C for 70 hours starting from freshly laid eggs. The number of animals analyzed is indicated on each bar. B. Morphological assessment shows that a subpopulation of animals carrying the maIs134 transgene become dauer larvae, which exhibit a lean body and darkened intestine (Figure S3). MYRF-1 overexpression (ybqIs112) suppresses the constitutive dauer formation in maIs134. Animals were cultured at 20°C for 70 hours starting from young L1. The number of animals analyzed is indicated on each bar. C. The development of maIs134 is delayed compared to wild-type animals. with the majority of maIs134 animals exhibiting pre-dauer-like characteristics while most of the wild-type animals become L4. While most of the wild-type animals become L4, the majority of maIs134 animals are pre-dauer-like. Animals were cultured at 20°C for 48 hours starting from freshly laid eggs. D. Representative images of animals from experiments in C. E. Measurements of body length of wild type and maIs134 animals show a growth delay in maIs134 starting from L2. The mean body length of analyzed animals at a series of time points is shown on the graph, with the mean ± S.D. indicated. The number of animals analyzed for each data point is more than 20. F. Representative images of animals from experiments in E. At 26h maIs134 animals are thinner than wild type and have dark intestinal granules, which are characteristic of pre-dauer (L2d). G. A tandem DNA array containing lin-4 promoter (2.4 kb) DNA causes puncta of GFP::MYRF-1(ybq14) in the nucleus. As a control, a 7xTetO sequence-containing DNA array causes the puncta formation of TetR::tagRFP(ybqSi233), while it doesn’t cause the aggregation of GFP::MYRF-1. Only the addition of lin-4 promoter DNA causes the formation of GFP::MYRF-1 puncta. H. Representative images of animal cells carrying transgenes described in G, but in a high magnification view. I. Line plots of signal intensity measurements along the bar ROI drawn across one red punctum in images, examples of which were shown in H. The bar ROI is centered at the fluorescent spot and examples of bar ROI were shown in H. Each individual panel represents signals from one cell.

MYRF-1 regulates a selective subset of microRNAs during L1-L2 transition.

A. The hierarchical clustering diagram shows differential miRNA expression levels in wild-type and myrf-1(ju1121) animals at L2 stage. The color scale represents the log2 of the fold change, with red indicating highly expressed miRNAs and blue indicating low-expressed miRNAs. The numbers indicate the mean TPM of the specific miRNA from three replicates. Significance was determined at P<0.05. B. The phylogenetic analysis shows the relationship between differentially expressed miRNA genes in myrf-1(ju1121). The three branches are color-coded. C. The expression of transcriptional reporters, Pmir-48-gfp(ybqSi206), Pmir-73-gfp(ybqSi208), and Pmir-230-gfp(ybqSi209) in wild-type and myrf-1(ju1121) animals at early-mid L1 (6h) and middle L2 (24h) is shown. D. The fluorescence intensity of transcriptional reporters shown in (C) is quantified and presented as mean ± SEM (t-test: ns, not significant, *p<0.05, ****p<0.0001); n >10, where n is the number of animals analyzed. E. An illustration for how MYRF is processed and promotes lin-4 expression during development. At early L1, MYRF is mainly localized to the cell membrane in a PAN-1 dependent manner. However, during late L1, N-MYRF is released from the membrane through the catalytic activity of the ICE (Intramolecular Chaperone of Endosialidase) domain following its trimerization. Subsequently, N-MYRF translocates to the nucleus to enhance the transcription of lin-4.

Plin-4-GFP expression in myrf-1 loss of function mutants.

A. Plin-4-GFP(maIs134) expression was examined in wild-type, myrf-1(syb1491), and myrf-1(syb1468) animals at the L2 stage (24h). Plin-4-GFP was not detected in myrf-1 mutants. B. The fluorescence intensity of Plin-4-GFP was quantified and presented as mean ± SEM (t-test; ****p<0.0001); n >30, where n is the number of animals analyzed. C. An individual of myrf-1(ju1121) mutant, showing DIC image and lin-4p::nls::mScarlet(umn84) fluorescence. mScarlet is expressed in 8-9 pharyngeal nuclei.

MYRF-1 is required for lin-4 transcription in all larval stages.

A. Larval stages L2, L3, and L4 animals were treated with or without 4 mM K-NAA for 12 h. The lin-4 transcription reporter was down-regulated in the drug-treated animals compared to the pre-treated and mock-treated animals. B. The fluorescence intensity of lin-4 transcription reporter was quantified and presented as mean ± SEM (t-test; ****p<0.0001); n >30, where n is the number of animals analyzed.

Constitutive dauer phenotype observed in a subpopulation of Plin-4-GFP(maIs134) animals.

Representative images of animals from the experiments in Figure 4B, which were cultured at 20°C for 70 hours, starting from synchronized young L1 larvae. A subset of animals carrying the maIs134 transgene exhibited a constitutive dauer phenotype even when food was not depleted on the culture plate.

lin-4 promoter DNA causes aggregation of GFP::MYRF-1(ybq14) in the nucleus.

A. Tandem DNA arrays containing lin-4 promoter (498 bp) DNA caused aggregation of GFP::MYRF-1(ybq14) in the nucleus (arrow), while a control co-marker (Pmyo-2-mCherry) containing DNA array did not cause the aggregation of GFP::MYRF-1. B. Tandem DNA arrays containing lin-4 promoter (2.4 kb) DNA caused aggregation of GFP::MYRF-1(ybq14) in the nucleus (arrow), while a control co-marker (Pmyo-2-mCherry) containing DNA array did not cause the aggregation of GFP::MYRF-1. C. Representative images of animal cells carrying transgenes described in B, but in a high magnification view. D. Line plots of signal intensity measurement along the bar ROI drawn across the GFP spots as illustrated in representative images in C. The bar ROI is centered at one fluorescent punctum. Each individual line represents signals from one representative cell. Examples of bar ROI were drawn on the images in C.