Role of PRY-1/Axin in heterochronic miRNA-mediated seam cell development

Background Caenorhabditis elegans seam cells serve as a good model to understand how genes and signaling pathways interact to control asymmetric cell fates. The stage-specific pattern of seam cell division is coordinated by a genetic network that includes WNT asymmetry pathway components WRM-1, LIT-1, and POP-1, as well as heterochronic microRNAs (miRNAs) and their downstream targets. Mutations in pry-1, a negative regulator of WNT signaling that belongs to the Axin family, were shown to cause seam cell defects; however, the mechanism of PRY-1 action and its interactions with miRNAs remain unclear. Results We found that pry-1 mutants in C. elegans exhibit seam cell, cuticle, and alae defects. To examine this further, a miRNA transcriptome analysis was carried out, which showed that let-7 (miR-48, miR-84, miR-241) and lin-4 (lin-4, miR-237) family members were upregulated in the absence of pry-1 function. Similar phenotypes and patterns of miRNA overexpression were also observed in C. briggsae pry-1 mutants, a species that is closely related to C. elegans. RNA interference-mediated silencing of wrm-1 and lit-1 in the C. elegans pry-1 mutants rescued the seam cell defect, whereas pop-1 silencing enhanced the phenotype, suggesting that all three proteins are likely important for PRY-1 function in seam cells. We also found that these miRNAs were overexpressed in pop-1 hypomorphic animals, suggesting that PRY-1 may be required for POP-1-mediated miRNA suppression. Analysis of the let-7 and lin-4-family heterochronic targets, lin-28 and hbl-1, showed that both genes were significantly downregulated in pry-1 mutants, and furthermore, lin-28 silencing reduced the number of seam cells in mutant animals. Conclusions Our results show that PRY-1 plays a conserved role to maintain normal expression of heterochronic miRNAs in nematodes. Furthermore, we demonstrated that PRY-1 acts upstream of the WNT asymmetry pathway components WRM-1, LIT-1, and POP-1, and miRNA target genes in seam cell development. Electronic supplementary material The online version of this article (10.1186/s12861-019-0197-5) contains supplementary material, which is available to authorized users.

, and lin-28 [10]. HBL-1 is a zinc-finger transcription factor that critically mediates embryogenesis [11], and that controls developmental timing during post-embryonic development [9]. LIN-14 is a novel class of transcription factor [12] that is initially expressed at high levels in hypodermal blast cells in newly hatched L1 animals but at lower levels by the L2 stage [13]. LIN-28 is a conserved RNA-binding protein that controls the maturation of let-7 miRNA [5,[14][15][16]. Hypomorphic and null alleles of lin-14 and hbl-1 cause an increase whereas lin-28 mutants cause a decrease in the overall number of seam cells [9,17,18].
In addition to heterochronic miRNAs and their targets, seam cell division is also regulated by the divergent WNT asymmetry pathway, whose components include the β-catenins WRM-1 and SYS-1, the NEMO-like kinase (NLK) LIT-1, and the T-cell factor/lymphoid enhancer factor (TCF/LEF) POP-1 [18,19]. Removal of POP-1 activity causes seam cells to divide symmetrically, and thereby leads to an increase in their number. Conversely, since LIT-1 normally forms a complex with WRM-1 to phosphorylate and thus stimulate POP-1 export from the nucleus, disrupting WRM-1 and/or LIT-1 activity reduces the number of seam cells [19]. Similarly, the ratio of nuclear POP-1/SYS-1 activity affects the fate of daughter cells, such that those with lower POP-1 (and hence comparatively higher SYS-1) levels retain their seam cell fate, whereas those with higher POP-1 (and hence comparatively lower SYS-1) levels differentiate [20][21][22][23]. Genetic studies have also shown that WNT asymmetry pathway components interact with heterochronic genes to control seam cell development [17,18].
While investigating the role of pry-1 in developmental and post-developmental processes, we observed that pry-1(mu38) animals exhibit weaker cuticle and abnormal alae. Further analysis revealed that they also display a higher number of seam cells, a phenotype that was previously reported [19]. Similar defects were also seen in a C. briggsae pry-1 mutant Cbr-pry-1(sy5353) [24], suggesting a conserved role for pry-1 in seam cell development and cuticular alae formation. These observations are in line with our recent pry-1(mu38) mRNA transcriptome profiling (RNA-Seq), which identified differentially expressed (DE) genes associated with 'cuticle development' [25]. Given that the heterochronic pathway involves both protein-coding and miRNA genes, in the present study we conducted an miRNA-specific whole genome RNA-Seq experiment, which uncovered six DE miRNAs that include members of lin-4 and let-7 families. To understand the interaction of pry-1 with miRNAs during seam cell development, we knocked-down WNT asymmetry pathway components. Reducing wrm-1 and lit-1 expression suppressed, while silencing pop-1 exacerbated the pry-1 phenotype. Furthermore, an miRNA expression analysis conducted in a pop-1 hypomorph revealed a similar upregulation of miR-NAs to that observed in pry-1(mu38) worms, suggesting both that POP-1 is critical for asymmetric seam cell division, and that its nuclear levels are likely reduced in pry-1 mutants. Overall, our data demonstrates the importance of PRY-1 and its interactions with the WNT asymmetry pathway components for the regulation of miRNAs (and their targets) during asymmetric cell division. Since the WNT pathway and miRNAs are conserved in eukaryotes, similar interactions with Axin family members may control stem cell division in other systems.

Results
We observed that pry-1 mutant worms have a weaker cuticle (Fig. 2a) and abnormal alae that frequently includes Fig. 1 Seam cell asymmetric divisions during postembryonic development in C. elegans. During postembryonic development, C. elegans larvae undergo a series of molts, each of which is associated with carefully timed seam cell divisions. In the V1-4, and V6, cells undergo stem cell-like division where the anterior daughter fuses with the hyp7 syncytium. The posterior daughter then self-renews as another 'stem cell-like' seam cell. The exception to the asymmetric divisions is in early L2 stage where they undergo one symmetrical division prior to dividing again in an asymmetric manner gaps (Fig. 2b, c). The phenotypic analysis of C. briggsae pry-1 mutants, Cbr-pry-1(sy5353) [24], revealed similar gaps in alae as well as defective seam cell morphologies (Fig. 2d-f). Given these hypodermis-associated phenotypes, we investigated the role of pry-1 in seam cell development.

pry-1 mutants exhibit an increased number of seam cells
In C. elegans, seam cells divide asymmetrically at each larval stage to produce two daughter cells, one of which fuses with the hypodermal syncytium, while the other retains the seam cell fate (Fig. 1). The L2 stage is unique (e, f) Seam cells in control AF16 (e) and Cbr-pry-1(sy5353) (f) are visualized by adherens-junction-associated marker Cel-dlg-1p::GFP. Cbr-pry-1(sy5353) animals show defects in cell fusion (scale bar 0.1 mm). Boxed areas, marked by dotted lines, have been enlarged in the second row. Scale bars in b, d-f are 0.01 mm. (g) Both the pry-1(mu38) and pry-1(gk3682) animals show increased seam cell numbers compared to control N2 (scale bar 0.1 mm). (h) Each control animal has exactly 16 seam cells, whereas an average of 21 and 19 cells are found in pry-1(mu38) and pry-1(gk3682) mutants, respectively. (i) pry-1(mu38) animals show increased seam cell number by the end of the L2 stage. In panels a, h, and i, each data point represents the mean of at least two replicates (each batch with 30 or more worms) and error bar represents the STD. Student's t-test was used to determine the statistical significance: *p < 0.05 because it also includes a symmetric division that causes an increase in the number of seam cells (Fig. 1). We found that the pry-1(mu38) mutants have an average of approximately five extra seam cells (Fig. 2g, h), consistent with a previously report [19]. A similar phenotype was also observed in pry-1(gk3682), a new CRISPR/Cas9-induced mutant strain (provided by Don Moerman's lab) (Fig. 2g, h; Additional file 1: Figure S1). A stage-specific analysis conducted using scm:: GFP and ajm-1::GFP markers [26] revealed a higher number of seam cells in pry-1 mutants by the end of the L2 stage, likely due to an increase in symmetric cell divisions (Fig. 2h, i). Together, these findings suggest that pry-1 appears to play a role in L2-specific seam cell division.
Heterochronic miRNA expression is altered in pry-1 mutants As described above, seam cell asymmetry is mediated by two interacting pathways. While heterochronic genes, such as members of the lin-4 and let-7 miRNA families, control cell division, the WNT asymmetry pathway plays a role in the specification of anterior/posterior daughter cell fates. To evaluate the involvement of miRNAs in pry-1-mediated seam cell development, we performed RNA-Seq experiment in L1-stage animals. The results revealed six DE miRNAs in the pry-1(mu38) mutants. Additionally, 61 novel miRNAs were recovered in the C. elegans reference sample (N2) (see Methods and Additional files 2-4: Table S1, S2, S3) that serve as a resource to further investigate the miRNA biology in worms.
To elucidate whether pry-1-mediated miRNA regulation is stage-specific, we examined miRNA transcript levels by qRT-PCR in adult nematodes. While the expression of miRNAs was either unchanged or downregulated in pry-1(mu38) (Additional file 6: Figure S3A), the pattern was different in Cbr-pry-1(sy5353) animals, i.e., three miRNA orthologs were up, two were down, and one was unchanged (Additional file 6: Figure S3B). We also used the existing C. elegans miRNA::GFP transgenic strains [29] to determine changes in miRNA expression in pry-1(mu38) animals and confirmed the qRT-PCR results (Additional file 7: Figure S4). Overall, the dissimilar expression trends of the analyzed miRNAs in adults from L1-stage nematodes suggests that the pry-1-miRNA network is likely temporally regulated. miR-246, which a previous study showed to be involved in aging, oxidative stress, and thermo-sensation [30,31], was the only miRNA to be downregulated in the pry-1(mu38) nematodes. Although no role in heterochronic development has yet been reported for miR-246, we herein demonstrate that miR-246(n4636) adults exhibit alae defects (Fig. 3e). Conversely, scm::GFP and ajm-1::GFP reporter-based expression analyses of the miR-246(n4636) adults did not reveal any significant change to the seam cells (Fig. 3f). This finding was supported by the results of the tissue-enrichment analysis (Additional file 5: Fig. S2). Interestingly, a hypodermal cell marker, dpy-7::H1-wcherry, revealed that the number of hypodermal cells was reduced in the mutant compared to control worms (45.1 ± 1.7, n = 20 and 51.9 ± 1.6, n = 20, respectively; also see Additional file 8: Figure S5). Further study is needed to determine the exact fate of these hypodermal cells in miR-246 mutants.
Many predicted gene targets of the mis-regulated heterochronic miRNAs are differentially expressed in the pry-1(mu38) mRNA transcriptome miRNAs mediate the degradation or translational inhibition of their target mRNAs via binding between their seed sequence and an miRNA response element (MRE) in the 3′ untranslated region of their target. Therefore, we searched for miRNA targets using TargetScan online program (http:// www.targetscan.org/vert_72/), and resultantly identified 453 unique targets. The gene ontology (GO) analysis revealed that these predicted miRNA targets were predominantly associated with processes such as the regulation of heterochrony (29-fold enrichment), the positive regulation of nematode larval development (8-fold enrichment), the molting cycle (5-fold enrichment), and collagen and cuticulinbased cuticle development (7-fold enrichment) (Fig. 4a).
A comparison of the predicted miRNA target genes with our recently published pry-1(mu38) mRNA transcriptome [25] revealed a significant overlap (111 genes, Representation factor: 1.6, hyp.geo p < 7.862 e-08) (Fig.  4b). Furthermore, a tissue-enrichment analysis showed that this subset of overlapping genes is frequently associated with the hypodermal syncytium (i.e. the third-most enriched subset) (Fig. 4c). Together, these findings suggest that PRY-1 is likely necessary for normal miRNA expression during seam cell development.
Knockdowns of WNT asymmetric pathway components affect both the pry-1(mu38) phenotype and miRNA expression We induced RNAi-mediated silencing to examine interactions between pry-1 and WNT asymmetry pathway components during seam cell development. The fates of seam cell daughters are specified by the nuclear levels of POP-1 that are high in the anterior cell (hypodermal fate) and low in the posterior cell (seam cell fate) [19,32] (Fig. 5a). The results of our experiments revealed that knockdowns of wrm-1 or lit-1 suppresses the pry-1(mu38) seam cell phenotype (Fig. 5b), likely because PRY-1 promotes asymmetric division by negatively regulating both of these factors in anterior daughter cells (Fig. 5a). This is consistent with PRY-1 being localized to the anterior cortex of dividing seam cells [23]. A similar genetic interaction between pry-1, wrm-1, and lit-1 was previously reported to occur during the asymmetric division of embryonic EMS cells [33].
In contrast to wrm-1 and lit-1, pop-1 RNAi exacerbated the pry-1(mu38) phenotype, resulting in a significant increase in the number of seam cells (35.9 ± 10.8 in pry-1(mu38); pop-1(RNAi), compared to 20.8 ± 2.1 in pry-1(mu38), and 23.5 ± 5.7 in pop-1(RNAi)) (Fig. 5b, c). We also examined nuclear POP-1 asymmetry following RNAi knockdown of pry-1 and found that it was disrupted (Fig. 5d). These results agree with nuclear POP-1 levels being differentially regulated by WRM-1 and LIT-1 to be higher in the anterior, and lower in the posterior daughter cell [19,32] (Fig. 5a) Together, our findings support that pop-1 likely limits the number of seam cells that are produced by promoting the asymmetric division of their precursors. Since the asymmetric localization of WRM-1, LIT-1, and POP-1 are known to mediate fate specification in presumptive seam cells, our results suggest that PRY-1 likely facilitates the maintenance of these asymmetric expression patterns.
Given that the nuclear POP-1 and SYS-1 ratio determines the fate of daughter cells and SYS-1 localization is disrupted in animals lacking PRY-1 function [34], we also examined the effect of sys-1 knockdown. The results of this analysis showed no effect on seam cell division in pry-1(mu38) animals (Fig. 5b). We likewise knocked down another β-Catenin family member, bar-1, in the mutant strain and found that doing so did not alter the number of seam cells (Fig. 5b). Overall, the data support the possibility that β-Catenin family members are functionally redundant during seam cell division, consistent with previous studies [19], although do not exclude the possibility that PRY-1 role in seam cells is independent of BAR-1 and SYS-1.
To examine whether WRM-1, LIT-1, and POP-1 asymmetries affect miRNA expression during seam cell division, we next quantified miRNA levels in animals in which POP-1 function was compromised. As in pry-1(mu38) mutants, the expression levels of lin-4, miR-48, miR-84, miR-237, and miR-241, were found to be significantly upregulated in both pop-1(hu9) and pop-1(RNAi) worms; however, no change to miR-246 expression was  [25]) and predicted targets of DE miRNAs identified by Targetscan (435 genes, this study) in pry-1(mu38) animals. Further analysis revealed that 435 potential targets are shared between DE miRNAs (36 by lin-4, 115 by miR-48, 115 by miR-84, 36 by miR-237, 115 by miR-241, and 102 by miR-246). A total 111 genes are common between the two data sets, a number that is statistically significant based on the hypergeometric test. (c) Tissue-enrichment analysis of the common set of genes (111) revealed third highest fold enrichment in hypodermal syncytium cells (colour coded in orange) observed (Fig. 5e, f). Furthermore, the bioinformatic analysis revealed multiple TCF/LEF consensus binding sites (SCTTTGATS; S = G/C) [35,36] in the 5′ regulatory region of each of these miRNAs, except for miR-246 where a single site is found near the transcriptional start site (See Methods, Additional file 9: Figure S6), suggesting that their transcription may be inhibited by POP-1. Together, these data allow us to conclude that miRNAs act downstream of POP-1, which agrees with a previous model [18], and also suggest that PRY-1 may interact with WRM-1, LIT-1, and POP-1 to negatively regulate the expression of heterochronic miRNAs during seam cell development.
The qRT-PCR analysis showed that while lin-14 expression levels were unchanged, hbl-1 and lin-28 were significantly downregulated in L1-stage pry-1(mu38) animals (Fig. 6a). Together with results described in previous sections, this observation allows us to propose that pry-1 may function upstream of miRNAs to promote expression of hbl-1 and lin-28. To further examine the regulatory network of pry-1, RNAi was carried out. The results revealed that while lin-14 and hbl-1 RNAi had no marked impact on seam cells, lin-28 RNAi caused a significant reduction in the seam cell number in both pry-1(mu38) and control animals (Fig. 6b).

Discussion
In this paper we describe a genetic pathway of PRY-1/ Axin signaling in seam cell development. Using a combination of mutant analysis and reporter gene expression we show that PRY-1 is involved in L2-specific seam cell division. To identify the genes regulated by pry-1, we performed whole genome miRNA profiling at the late-L1 stage. The results revealed six DE miRNAs in pry-1 mutants. Five of these, belonging to lin-4 and let-7 families (lin-4, miR-48, miR-84, miR-237, and miR-241), were upregulated whereas miR-246 was the only miRNA that was downregulated. A similar trend was also observed in C. briggsae pry-1 mutants suggesting that pry-1 plays a conserved role in miRNA regulation in Caenorhabditis nematodes.
Three of the overexpressed miRNAs in pry-1 mutants, namely miR-48, miR-84, and miR-241 (let-7 family members), are known to redundantly control the L2-L3 larval-stage transition [9]. While C. elegans nematodes carrying a mutation in any one of these three miRNAs have been shown to exhibit a normal phenotype, miR-48/miR-84 double mutants display retarded molting and a higher number of seam cells as a result of reiterated symmetric divisions during the L2 stage [9]. This seam cell phenotype is further exacerbated in miR-48/miR-84/ miR-241 triple mutants [9], while conversely, miR-48overexpression mutants were shown to exhibit a reduced number of seam cells due to 'skipping' of L2-specific symmetric divisions [41]. The other two miRNAs upregulated in the pry-1(mu38) mutants are lin-4 and miR-237 (lin-4 family members [42]). A previous study showed that, although a miR-237-knock-out mutant does not directly incur a heterochronic defect, it enhances the seam cell phenotype exhibited by lin-4(e912); lin-14(n179ts) double mutant animals [8].
Unlike lin-4 and let-7 family of DE miRNAs, miR-246 is not known to play a role in heterochronic development although it is involved in other processes [30,31]. Consistent with previous studies, our analysis of miR-246 mutants did not reveal any changes in the number of seam cells. Thus, pry-1-mediated miR-246 regulation may participate in other biological events.
The fact that all but one DE miRNAs in pry-1 mutants are involved in heterochronic pathway suggests an important role of pry-1 in this developmental process. This was further strengthened by our data showing a high enrichment for miRNA predicted target genes associated with GO-term processes such as regulation of heterochrony. Moreover, we observed a significant overlap between DE miRNA predicted targets and pry-1(mu38) mRNA transcriptome [25] that included many genes expressed in hypodermal syncytium.
To understand the regulation of miRNAs by pry-1 we studied the involvement of WNT asymmetry pathway components using an RNAi approach. It was shown Fig. 6 Analysis of heterochronic genes in pry-1-mediated seam cell development. (a) qRT-PCR experiments at the L1 stage showed that hbl-1 and lin-28 are significantly downregulated in pry-1(mu38) animals. Each data point represents the mean of two replicates and error bar represents the SEM, **p < 0.01. (b) RNAi KD of lin-28 in pry-1(mu38) mutants rescued the seam cell defect. Each data point represents the mean of three batches (each batch with at least 30 worms) and error bar represents the STD. Student's t-test was used to determine the statistical significance: *p < 0.05 (compared to L4440 control) earlier that in the absence of PRY-1, localizations of WRM-1, LIT-1, and SYS-1 are disrupted [23,34]. As expected, the examination of seam cell phenotypes in pry-1(mu38) animals following RNAi knockdowns of these genes revealed that wrm-1 and lit-1 are necessary for pry-1 function. Thus, PRY-1 may affect seam cell number by localizing in the anterior cortex of dividing seam cells [23] and thereby lowering WRM-1 and LIT-1mediated nuclear POP-1 levels in anterior daughters. This plausible explanation is supported by our finding that POP-1 asymmetry is affected in animals with reduced PRY-1 function. To test this further, we examined miRNA expression in pop-1(hu9) and pop-1(RNAi) worms. As expected, all five (lin-4, miR-48, miR-84, miR-237, and miR-241) were found to be overexpressed. Moreover, multiple TCF/LEF binding sites were detected in the 5′ regulatory regions of miRNAs. Together, these findings raise the possibility of POP-1 acting as a transcriptional regulator of miRNAs.
We thus propose a model summarizing our findings (Fig. 7), in which PRY-1 acts upstream of WRM-1, LIT-1, POP-1, and DE miRNAs (except miR-246). Since the miRNAs regulate the expression of protein-coding genes during heterochronic development, we tested three of their known targets hbl-1, lin-14, and lin-28. The results of our experiments suggested that hbl-1 and lin-28 act downstream of pry-1; however, only lin-28 appears to function in pry-1-mediated asymmetric cell division. The above genetic interactions are consistent with earlier findings where lin-28 was shown to act downstream of the WNT asymmetry pathway components and included in a network consisting of lin-4 and let-7-family of miR-NAs and their targets during seam cell development [17,18]. Our model is unique in that it places PRY-1 upstream of WRM-1, LIT-1, and POP-1-mediated miRNA transcriptional network.

Conclusions
Overall, the data presented in this paper demonstrate the important role of PRY-1/Axin in the regulation of miR-NAs and their heterochronic gene targets in a pathway that involves WNT asymmetry pathway components WRM-1, LIT-1, and POP-1 during seam cell development. Furthermore, since seam cell defects are also exhibited by C. briggsae pry-1 mutants, and that Cbr-pry-1 is necessary for the normal expression of these miRNA orthologs, our work has revealed that the role of pry-1 in seam cell development is conserved amongst nematodes.

Strains, culture condition, and RNAi
Nematodes were grown on standard NG-agar culture plates seeded with E. coli bacteria (OP50) [43]. Cultures were maintained at 20°C. Strains used in the study are listed in a supplementary table (Additional file 10: Table S4). RNAimediated gene silencing was performed using a protocol previously published by our laboratory [24].

Microscopy
Nematodes were mounted on a glass slide containing 2% agarose and 0.02 M NaN 3 and observed using an Axiovision Zeiss microscope. Seam cell nuclei were counted, and adult lateral alae were scored using Nomarski differential interference contrast and epifluorescence optics. Images were acquired using NIS Element software (Nikon, USA) with a Hamamastu Camera that was mounted on a Nikon 80i upright microscope.

Analysis of seam cell division
The fates of daughter seam cells were determined in C. elegans using scm::GFP and ajm-1::GFP markers, and in C. briggsae using the seam cell adherens-junction marker, Cel-dlg-1::GFP. After 8 h of feeding, nematodes expressing GFP that had completed the first larval seam cell division, and that had 10 seam cells per side, were chosen for analysis. Seam cell divisions were monitored at approximately 6 h intervals until the late L4 stage, when divisions ceased.

Cuticle integrity assay
A solution containing 1% hypochlorite and 0.25 M NaOH was prepared as previously reported [44], and aliquoted (500 μl) into a 48-well plate. Individual gravid Fig. 7 A model summarizing genetic interactions between PRY-1, WNT asymmetric pathway components (WRM-1, LIT-1, and POP-1), heterochronic miRNAs, and the targets of heterochronic miRNAs during L2 stage seam cell development. Our data supports the Ren and Zhang model [18] and places the WNT asymmetric pathway upstream of miRNAs adult worms were transferred to each well, and the plate was agitated at 30 s intervals. The time to the first major cuticle break was recorded by direct observation using a dissecting stereoscope (SMZ 645; Nikon Corporation, Japan).

Molecular biology and bioinformatics
For qRT-PCR experiments, synchronous cultures were prepared by bleaching gravid hermaphrodites as described previously [45] except that eggs were allowed to hatch on the NG-agar plates. The bleaching process was repeated. The eggs were finally transferred onto plates and grown till the desired stage. Because pry-1 mutants grow slower than controls, RNA was extracted from age-matched animals. The L1 larvae were grown till 16 h (N2/AF16) and 18 h (pry-1 mutants) whereas adults were incubated for 52 h (N2/AF16) and 58 h (pry-1 mutant). Total RNA was extracted from animals using the trizol reagent (Catalog Number T9424, Sigma-Aldrich, Canada), according to the manufacturer's instructions. cDNAs for protein-encoding genes and miRNAs were synthesized using oligo-dT and specific stem-loop primers, respectively (Additional file 11: Table S5), and by using the qScript cDNA synthesis kit (Catalog Number 95047-025, Quantabio, Canada) according to manufacturer's instructions.
qRT-PCR was performed (in triplicate) in the Bio-Rad cycler CFX 96 using appropriate primers (Additional file 11: Table S5), and SensiFAST SYBR Green Kit (Catalog Number BIO-98005, BIOLINE, USA), according to the manufacturer's instructions. The expression levels of miR-NAs and protein-coding genes were normalized to those of miR-2 and pmp-3, respectively. Ct and p values were calculated using CFX manager software (Bio-Rad, Canada).
A lin-28 RNAi plasmid was constructed by inserting 2949 bp of the lin-28 coding sequence into the L4440 vector. A DNA fragment was obtained via PCR using the listed primers (Additional file 11: Table S5), under the specified PCR conditions.
To identify TCF/LEF family binding sites in the 5′ upstream genomic region of the miRNAs, MatInspector software (https://www.genomatix.de/) was used with default settings.

RNA-Seq experiment
The steps for miRNA RNA-Seq in pry-1(mu38) mutants were similar to mRNA RNA-Seq that we reported earlier [25]. The pry-1 miRNA transcriptome profile can be found in the GEO archive with accession number GSE130039. Synchronized L1 worms were harvested by two successive bleaching to obtain a homogenous population and total RNA was isolated. Small RNA sequencing libraries were prepared, and samples were analysed using the Genome Analyzer IIx platform (Illumina Inc., USA) at the McGill University Genome Quebec sequencing facility.
A total of 36,656,022 reads of small RNAs (15-25 nt) were generated from the four samples of C. elegans examined, of which 10,453,527 sequences aligned perfectly to the C. elegans genome. Overall, we detected perfect matches to the precursor forms of 161 out of the 250 miRNAs annotated in miRBase (http://www.mirbase.org) WBcel235 for C. elegans. DE analysis of the known miR-NAs led to identification of eight miRNAs that were altered in pry-1 mutants, of which two (miR-353 and miR-2208a) were excluded due to false predictions (Additional file 2: Table S1).
Although previous studies have reported miRNAs in the C. elegans genome (e.g., see [42,46], due to increased depth of our sequencing data we expected to uncover additional new candidates. After eliminating rRNA, tRNA, and ncRNA reads, the remaining unannotated reads were processed for novel miRNA discovery, as discussed below. We focused on the 30,300 nonredundant un-annotated reads that aligned to the C. elegans genome in control N2 and pry-1(mu38) animals. To discover novel miRNAs, the miRNA discovery package miRDeep2 (https://www.mdc-berlin.de/n-rajews-ky#t-data,software&resources) was used. The analysis predicted a total of 243 miRNAs using read count cutoff of 5-fold (or 187 with cut-off of 10-fold) (Additional file 3: Table S2). We then used an additional criterion to further examine these candidates, i.e., a higher miRDeep Score (> 10). This led to the identification of 64 novel miRNAs at the 5-fold read count cut-off (or 61 when the read count was set at 10-fold) (Additional file 3-4: Table S2, S3). The authenticity of these novel miRNAs was tested by RNAfold, which confirmed that these produce miRNA stem loop structure [47].

Statistical analyses
Statistics were performed by two-tailed student's t test after testing for equal distribution of the data and equal variances within the data set. The p values of 0.05 and less were considered statistically significant. The data are presented as either mean ± standard deviation of the mean (STD) or mean ± standard error of mean (SEM). Graphs were prepared using Microsoft Excel. Hypergeometric probability related tests were done using an online program (http://nemates.org/MA/progs/overlap_stats.html).

Additional files
Additional file 1: Figure S1. C. elegans pry-1 open reading frame showing the region affected by gk3682 mutation. The exons and introns are indicated by boxes and lines, respectively. The translational start and stop sites are marked. The sequence deleted in gk3682 allele (738 bp) is shown by a rectangle. As part of the CRISPR editing process, the excised