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Cell-cell interactions and cross-talk between signaling pathways specify Caenorhabditis elegans vulval precursor cells (VPCs) to adopt a spatial pattern: a central “1°” VPC, in which epidermal growth factor receptor (EGFR)–mitogen-activated protein kinase (MAPK) activity is high and LIN-12/Notch activity is low, flanked by two “2°” VPCs, in which LIN-12/Notch activity is high and EGFR-MAPK activity is low. Here, we identify a microRNA gene, mir-61, as a direct transcriptional target of LIN-12 and show that expression of mir-61 promotes the 2° fate. We also identify vav-1, the ortholog of the Vav oncogene, as a target of mir-61, and show that down-regulation of VAV-1 promotes lin-12 activity in specifying the 2° fate. Our results suggest that lin-12, mir-61, and vav-1 form a feedback loop that helps maximize lin-12 activity in the presumptive 2° VPCs.
Six multipotential VPCs, numbered P3.p to P8.p, adopt an invariant pattern of fates termed 3°-3°-2°-1°-2°-3° (Fig. 1A). Two signaling events specify this pattern: “inductive” signaling, mediated by an EGFR-Ras-MAPK pathway, and “lateral” signaling, mediated by LIN-12 (Fig. 1A) (1). The inductive signal from the gonad activates an EGFR-Ras-MAPK cascade in a graded fashion in the underlying VPCs, P5.p, P6.p, and P7.p. The centralmost VPC, P6.p, has the highest level of EGFR-Ras-MAPK activation and becomes the presumptive 1° VPC; it produces the lateral signal, which activates LIN-12 in P5.p and P7.p. When LIN-12 is activated, proteolysis releases its intracellular domain, which translocates to the nucleus and forms a transcriptional activation complex with the DNA binding protein LAG-1 (2). Transcriptional targets of LIN-12 in P5.p and P7.p can be identified by the presence of LAG-1 binding sites (LBSs) in their 5′ flanking regions and include genes that encode negative regulators of EGFR-Ras-MAPK activity in P5.p and P7.p, which inhibit the expression of 1° fate features in these cells (3).
Short regulatory microRNAs (miRNAs), first identified in C. elegans (4), mediate posttranscriptional down-regulation of target genes. The profound and pervasive roles that miRNAs play as critical regulators of developmental gene expression are only now becoming fully appreciated. We obtained an indication that a miRNA may be involved in lateral signaling after observing a lateral signaling defect when a miRNA-processing gene was depleted (5). We then computationally identified mir-61 as a potential miRNA that is transcribed when the lateral signal from P6.p activates LIN-12 in P5.p and P7.p (5). A mir-61 transcriptional reporter is specifically expressed in P5.p and P7.p and their daughters (Fig. 1B) (6), consistent with a function for mir-61 as a direct target of LIN-12 during lateral signaling. This inference was confirmed by mutating the LBSs in the mir-61 promoter and finding that expression in P5.p and P7.p was lost (Fig. 1C) (7).
Ectopic expression has been a successful approach to elucidating the role of miRNAs for which null alleles are not available and obviates potential problems that may be posed by functional redundancy (8-10). Expression of mir-61 ectopically in P6.p, the presumptive 1° VPC, causes expression of the canonical 2° fate marker lin-11::gfp, whereas expression of an unrelated miRNA does not (Fig. 2). These observations suggest that mir-61 activity promotes the 2° fate.
miRNAs bind to sites in 3′ untranslated regions (UTRs) of target mRNAs and inhibit their translation (8). Thus, we hypothesized that mir-61 is expressed in presumptive 2° VPCs in order to down-regulate potential target gene products that would interfere with specification of the 2° fate. We identified potential target genes of mir-61 computationally (5), requiring that 3′ UTRs have at least seven bases of perfect complementarity to the 5′ end of mir-61 and that the binding sites be conserved in C. briggsae orthologs. This analysis yielded three candidates, vav-1, inx-1, and egl-46 (Fig. 3A) (5).
To assess candidate genes in vivo, we developed a simple heterologous assay to circumvent potential detection problems due to weak or transient mir-61 expression in the VPCs. This assay may be used to test whether any miRNA can target the 3′ UTR of any candidate target gene. We expressed mir-61 in coelomocytes, distinctive cells for which strong promoters are available (11, 12). On a second transgene, we expressed two reporters in coelomocytes: one a yellow fluorescent protein (YFP) reporter with the 3′ UTR of the putative target gene and the other a cyan fluorescent protein (CFP) reporter with the unc-54 3′ UTR, which does not contain any mir-61–binding sites (13). If a candidate is a bona fide target, then we would expect to see coelomocytes displaying CFP expression and down-regulation of YFP expression. Using this assay, we obtained evidence that mir-61 can regulate the expression of the three candidate genes identified by using the criteria described above (Fig. 3A) (5).
mir-61 is also expressed in cells other than the VPCs, and lin-12 activity specifies many other cell fate decisions (14), so even bona fide targets of mir-61 may not be relevant to lateral signaling. The desired target genes should be transcribed in the VPCs but posttranscriptionally down-regulated by way of their 3′ UTRs in P5.p and P7.p and their daughters. We fused the 5′ upstream sequence of vav-1, inx-1, or egl-46 to yfp::unc-54 3′UTR and found that only vav-1 is expressed in the VPCs and their daughters (Fig. 3B). When we replaced the unc-54 3′UTR with the vav-1 3′UTR, creating a sensor construct, vav-1 expression was lost in P5.p and P7.p in a significant proportion of hermaphrodites; this loss depends on an intact mir-61 target site (Fig. 3B). These observations indicate that vav-1 is posttranscriptionally regulated in P5.p and P7.p, consistent with regulation by endogenous mir-61, and suggest that VAV-1 may be down-regulated in presumptive 2° VPCs to promote lin-12 activity.
VAV-1 is an ortholog of the Vav oncoprotein, which has guanine nucleotide exchange factor (GEF) activity and additional domains that mediate interactions with other proteins (15); thus, there are many different potential mechanisms by which Vav proteins may modulate the activity of signaling pathways in presumptive 2° VPCs. In mammalian cells, in some contexts, Vav appears to be a positive regulator of MAPK signaling, but in others, it has no effect (15). Loss of vav-1 activity does not prevent vulval induction, which indicates that vav-1 is not required for EGFR-MAPK signaling in the cellular context of VPCs (16) and that down-regulation of VAV-1 by mir-61 may not specifically attenuate EGFR-MAPK signaling in presumptive 2° VPCs in response to the low level of inductive signal (3).
Alternatively, VAV-1 may be a negative regulator of LIN-12. If so, then down-regulation of VAV-1 by mir-61 would increase lin-12 activity in P5.p and P7.p, independent of any input from the inductive signaling pathway. We therefore looked at whether loss of vav-1 activity enhances lin-12 activity under conditions where inductive signaling does not occur. The alleles lin-12(n379) and lin-12(n676) result in mild constitutive activity: Hermaphrodites lack an anchor cell, and all VPCs generally adopt the 3° fate, as there is no inductive signal to specify a 1° fate and insufficient constitutive lin-12 activity to promote anchor cell–independent 2° fates (14). In such backgrounds, loss of some negative regulators of LIN-12 increases LIN-12 stability or activity, which causes all six VPCs to adopt the 2° fate and to generate multiple pseudovulvae (17). Negative regulators that behave in this manner include SEL-10/Fbw7, which promotes ubiquitin-mediated turnover of LIN-12/Notch (18), and SEL-9, which functions in secretory protein quality control (19). We found that vav-1(RNAi) significantly enhances the constitutive activity of lin-12(n379) and lin-12(n676), which increases the number of hermaphrodites with multiple pseudovulvae (Fig. 4A). These results suggest that vav-1 is a negative regulator of lin-12 activity.
We have shown that mir-61 is a direct transcriptional target of the LIN-12/Notch pathway and that vav-1 is a target of mir-61 in the VPCs. We have also shown that ectopic mir-61 promotes the 2° fate and that VAV-1 is a negative regulator of lin-12 activity. We propose that activation of mir-61 transcription by LIN-12 and the consequent down-regulation of VAV-1 constitute a positive-feedback loop that promotes LIN-12 activity in presumptive 2° VPCs (Fig. 4B).
Although there are many possible molecular mechanisms that may underlie this positive-feedback loop, it is notable that Vav has many domains that could couple it to receptors or to mediators of endocytosis (15). Indeed, Vav proteins have recently been shown to affect endocytosis of the activated Ephrin receptor (20). Perhaps down-regulation of VAV-1 in presumptive 2° VPCs decreases the rate of internalization, promotes endocytic recycling of LIN-12 or required proteases, or alters another aspect of trafficking that favors ectodomain shedding or transmembrane cleavage.
Fig. S1. Lateral signaling defect in alg-1(RNAi) hermaphrodites.
Fig. S2. 5′ flanking regions of C. elegans and C. briggsae mir-61. The 5′ flanking regions of mir-61 in both species are predicted to be about 1 kb; the 5′ flanking region is defined as the expanse from the next predicted gene to the start of the mir-61precursor. The positions of conserved LBSs are shown with respect to the start of the coding region for the mir-61 precursor, symbolized by a hairpin.
Fig. S3. Alignment of C. elegans and C. briggsae mir-61.
Fig. S4. mir-61 targets. All were verified using the assay described in Fig. 3; however, only vav-1 appears to be expressed in VPCs.
Fig. S5. 3′ UTRs that do not permit downregulation by mir-61 in the coelomocyte assay. The unc-54 3′UTR also does not permit downregulation (see text and Fig. 3A).
We are grateful to X. Zhou for performing microinjections; and to O. Hobert, R. Mann, and D. Shaye for discussion and insightful comments on the manuscript. This work was supported by NIH grant CA095389 (to I.G.). I.G. is an investigator with the Howard Hughes Medical Institute.
Supporting Online Material www.sciencemag.org/cgi/content/full/1119481/DC1 Materials and Methods SOM Text Figs. S1 to S5 References and Notes