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Function of the PHA-4/FOXA transcription factor during C. elegans post-embryonic development Chen, Di; Riddle, Donald L Feb 29, 2008

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ralssBioMed CentBMC Developmental BiologyOpen AcceResearch articleFunction of the PHA-4/FOXA transcription factor during C. elegans post-embryonic developmentDi Chen1,2,3 and Donald L Riddle*1,2Address: 1Division of Biological Sciences, University of Missouri, Columbia, MO 65211, USA, 2Michael Smith Laboratories, University of British Columbia, Vancouver, BC V6T 1Z4, Canada and 3Buck Institute for Age Research, 8001 Redwood Blvd, Novato, CA 94945, USAEmail: Di Chen - dchen@buckinstitute.org; Donald L Riddle* - driddle@interchange.ubc.ca* Corresponding author    AbstractBackground: pha-4 encodes a forkhead box (FOX) A transcription factor serving as the C. eleganspharynx organ identity factor during embryogenesis. Using Serial Analysis of Gene Expression(SAGE), comparison of gene expression profiles between growing stages animals and long-lived,developmentally diapaused dauer larvae revealed that pha-4 transcription is increased in the dauerstage.Results: Knocking down pha-4 expression by RNAi during post-embryonic development showedthat PHA-4 is essential for dauer recovery, gonad and vulva development. daf-16, which encodes aFOXO transcription factor regulated by insulin/IGF-1 signaling, shows overlapping expressionpatterns and a loss-of-function post-embryonic phenotype similar to that of pha-4 during dauerrecovery. pha-4 RNAi and daf-16 mutations have additive effects on dauer recovery, suggestingthese two regulators may function in parallel pathways. Gene expression studies using RT-PCR andGFP reporters showed that pha-4 transcription is elevated under starvation, and a conservedforkhead transcription factor binding site in the second intron of pha-4 is important for theneuronal expression. The vulval transcription of lag-2, which encodes a ligand for the LIN-12/Notchlateral signaling pathway, is inhibited by pha-4 RNAi, indicating that LAG-2 functions downstreamof PHA-4 in vulva development.Conclusion: Analysis of PHA-4 during post-embryonic development revealed previouslyunsuspected functions for this important transcriptional regulator in dauer recovery, and may helpexplain the network of transcriptional control integrating organogenesis with the decision betweengrowth and developmental arrest at the dauer entry and exit stages.BackgroundAt the second larval molt, C. elegans may arrest develop-ment at the dauer stage in response to starvation and over-crowding, but can resume development to the adult whenan environment favoring growth is encountered [1]. Entryconstitutively released, dauer-inducing pheromone [2-4].Genes involved in dauer formation are called daf genes.Dauer-constitutive (Daf-c) mutants form dauer larvae inan environment with abundant food, whereas dauer-defective (Daf-d) mutants fail to enter the dauer stagePublished: 29 February 2008BMC Developmental Biology 2008, 8:26 doi:10.1186/1471-213X-8-26Received: 18 October 2007Accepted: 29 February 2008This article is available from: http://www.biomedcentral.com/1471-213X/8/26© 2008 Chen and Riddle; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.Page 1 of 10(page number not for citation purposes)into and exit from the dauer stage are determined by envi-ronmental cues, such as temperature, food supply and awhen they are starved or overcrowded. Most Daf-cBMC Developmental Biology 2008, 8:26 http://www.biomedcentral.com/1471-213X/8/26mutants are temperature-sensitive, revealing the naturaltemperature dependence of dauer formation [5].Many daf genes have been ordered in a branched geneticpathway based on genetic epistasis [5]. Three functionallyoverlapping pathways, including TGF-β, insulin/IGF-1,and cyclic GMP signaling pathways, are involved inresponding to environmental cues [6]. DAF-7, a memberof the TGF-β superfamily of protein growth factors, signalsthrough downstream receptor kinases, SMAD transcrip-tion factors and the DAF-12 nuclear hormone receptor toinhibit dauer formation and promote reproductive devel-opment [7-12]. The daf-2 gene encodes an IGF-1 receptor,which functions through downstream kinases to phos-phorylate the DAF-16/FOXO transcription factor [13-17],and to influence the biosynthesis of ligands for DAF-12[18-21].As a step toward understanding the genetic basis of dia-pause and longevity, Jones et al. [22] compared geneexpression profiles of dauer larvae and mixed-stage, grow-ing populations by SAGE. Transcripts that were enrichedeither in dauer larvae or in mixed stages were identified[22]. SAGE tags corresponding to pha-4, which encodes aFOXA transcription factor homolog, were detected in thedauer stage (8 tags), but not in mixed stages (no tags).PHA-4 is regarded as the organ identity factor for the C.elegans pharynx [23-25]. In pha-4 mutants, pharyngealcells are transformed into ectoderm and the developmentof mutant animals is arrested after hatching [25]. pha-4mRNA is highly enriched in both the pharyngeal andintestinal primordia of the embryo, and low levels of pha-4 transcripts can be detected in the L3/L4 larval somaticgonad [26]. Candidate PHA-4 target genes in pharyngealdevelopment have been identified [27], and analysis oftarget promoter sequences revealed several cis-regulatoryelements. These elements are targets of unknown tran-scription factors, which function coordinately with PHA-4 to modulate gene expression in different pharyngeal celltypes and at different developmental stages [28,29]. Ao etal. [28] found that the DAF-12 nuclear hormone receptoris one of these transcription factors. DAF-12 and PHA-4function together to either activate or inhibit myo-2, thepharyngeal muscle gene, in response to environmentaland developmental cues [28]. Thus, PHA-4 is an impor-tant regulator that is expressed in several cell types andcontrols a wide range of gene expression.To assess PHA-4 functions in dauer larvae, we treated Daf-c mutants with pha-4 RNAi. We found that PHA-4 is essen-tial for dauer recovery, gonad and vulva development. Thehermaphrodite somatic gonad includes distal tip cells,mitosis to meiosis in germline cells through the GLP-1/Notch signaling pathway [30]. Gonadal sheath cells havephysical contacts with the germline; the muscle compo-nents convey contractile properties required for ovula-tion. Moreover, the sheath cells, spermathecal and uterineprecursor cells play regulatory roles in germline develop-ment, such as promoting germline proliferation, exit frompachytene, and/or gametogenesis [31]. Identification ofPHA-4 targets in the somatic gonad should help explainthe PHA-4 gonadal phenotype. The vulval transcription oflag-2, which encodes a ligand for the LIN-12/Notch lateralsignaling pathway, is inhibited by pha-4 RNAi, indicatingthat LAG-2 functions downstream of PHA-4 in vulvadevelopment.ResultsPHA-4 is required for dauer recovery, gonad and vulva developmentSAGE revealed that pha-4 transcription is elevated in dauerlarvae [22], suggesting that, in addition to pharyngeal org-anogenesis, PHA-4 may function in dauer formation orrecovery. We used semi-quantitative RT-PCR to comparepha-4 transcript levels in mixed-stages, dauer, and at dif-ferent times during dauer recovery. pha-4 transcripts weremuch more abundant in the dauer stage than in mixedstages, and were also detected shortly after dauer larvaestarted to recover. pha-4 transcripts decreased duringresumption of development (Fig. 1).pha-4 transcripts are enriched in dauer larvaeFigure 1pha-4 transcripts are enriched in dauer larvae. Semi-quantitative RT-PCR experiments were performed to deter-mine levels of pha-4 and rpl-21 mRNA. rpl-21, which encodes a ribosomal protein, serves as the internal control for equal loading. Experiments were performed three times with con-sistent results using three independent RNA preparations. Lane 1, mixed stages; lane 2, dauer; lane 3, 4 and 5, 2 hours, 24 hours and 36 hours after placement in food, respectively. Dauer larvae begin to feed approximately three hours after Page 2 of 10(page number not for citation purposes)gonadal sheath cells, spermathecae and uterus. Distal tipcells regulate gonadal arm elongation and the switch fromexposure to food.BMC Developmental Biology 2008, 8:26 http://www.biomedcentral.com/1471-213X/8/26To determine PHA-4 function in the dauer stage, weknocked-down pha-4 expression by feeding Daf-c mutantanimals E. coli expressing pha-4 double-stranded RNA[32] from the time of hatching from the egg. As a control,we fed the same strains with E. coli that carries the RNAivector (L4440) without any insert. Semi-quantitative RT-PCR showed that pha-4 transcripts were efficientlyreduced by the RNAi treatment (Fig. 2A).Dauer larvae with pha-4 RNAi treatment showed normaldauer characteristics, including SDS resistance and a con-stricted pharynx (not shown), suggesting that PHA-4 isnot required for dauer formation. However, when thedauer larvae were transferred to an environment favoringgrowth (fresh food and low temperature), they exhibiteddecreased recovery compared to the control, 2 – 32% vs.96 – 99% (Fig. 2B, Table 1). pha-4 RNAi-treated animalsthat resumed development to the adult had abnormaloocytes and embryos, and protruding vulvae (Fig. 2D,2F). Adults fed the RNAi control under the same condi-tions were normal (Fig. 2C, 2E). Thus, PHA-4 plays essen-tial roles in dauer recovery and gonad/vulva development.To assess the possible role of PHA-4 in gonad and vulvadevelopment, a pha-4::gfp translational fusion carrying the1.5 kb promoter (the first two exons and introns, and partof the third exon of pha-4 fused in-frame with the gfp cod-ing region) was injected into the germline of daf-2(e1370). GFP was present in distal tip cells (DTCs), intes-tinal cells (Fig. 3A) and neurons (not shown) in dauer lar-vae, and in the spermathecae and uteri of L4 larvae (Fig.3B).PHA-4 is required for dauer recovery, gonad and vulva developmentFigure 2PHA-4 is required for dauer recovery, gonad and vulva development. (A) pha-4 RNAi treatment efficiently inhibits pha-4 transcription. Semi-quantitative RT-PCR shows pha-4 and rpl-21 transcription levels in daf-7(e1372) and daf-2(e1370) dauer larvae treated with the RNAi control (lanes 1, 3) and pha-4 RNAi (lanes 2, 4). RT-PCR experiments were performed three times with consistent results using three independent RNA preparations. (B) PHA-4 is needed for dauer recovery. Dauer larvae formed constitutively at 25°C in the presence of pha-4 RNAi from hatching showed significantly decreased recov-ery upon downshift to 15°C compared to control daf-7(e1372), daf-1(m40), daf-2(e1370), and age-1(m865) mutants. Percent-ages of dauer recovery, numbers of animals scored, and p values for t-tests were shown in Table 1. The entire experiment was performed twice with triplicates for each treatment. (C-F) pha-4 is involved in gonad and vulva development. (C) Control post-dauer daf-7(e1372) adult; (D) pha-4 RNAi-treated post-dauer daf-7(e1372) adult; (E) control post-dauer daf-2(e1370) Page 3 of 10(page number not for citation purposes)adult; and (F) pha-4 RNAi-treated post-dauer daf-2(e1370) adult. The Daf-c mutants all exhibit similar gonad and vulva defects with variable expressivity. Anterior is left and dorsal is up. dtc, distal tip cell; germ, germline; oo, oocytes; emb, embryos; vul, vulva. Scale bars, 10 μm.BMC Developmental Biology 2008, 8:26 http://www.biomedcentral.com/1471-213X/8/26DAF-16 is also important for dauer recovery, gonad and vulva developmentPrevious studies showed that DAF-16B, one of the daf-16gene products, is expressed in the pharynx, somatic gonadand tail neurons [33]. The overlapping expression pat-terns of daf-16b and pha-4 and the essential roles of DAF-16 during dauer development suggest the possibility thatDAF-16 may be involved in dauer recovery. Since daf-16 isfrom the dauer stage. The daf-16(mgDf47) null mutantcarries the daf-16 mutation lacking the coding sequencefor DNA binding domains [33]. The daf-7 Daf-c mutantaffects the TGF-β pathway, and is not suppressed by daf-16mutations [34]. The daf-16; daf-7 dauer larvae formedconstitutively at 25°C showed significantly decreasedrecovery upon downshift to 15°C compared to the daf-7(e1372) dauer larvae (Fig. 4A, Table 1). Animals, whichresumed development to the adult, showed defects in thegonad and vulva similar to pha-4 RNAi-treated animals(Fig. 4B, 4C).DAF-16 and PHA-4 function in parallel pathways to regulate dauer recoverySince pha-4 transcripts are elevated in dauer larvae, tran-scription factors in dauer signaling pathways are candi-dates for promoting pha-4 expression in dauer larvae. Theoverlapping expression patterns and similar loss-of-func-tion phenotypes of daf-16 and pha-4 suggest the possibil-ity that DAF-16 may be a regulator of pha-4 transcription.pha-4 is transcribed into three major transcripts: pha-4a, band c [26]. pha-4 b and c are trans-spliced with the SL1leader mRNA at the beginning of exons 2 and 3, respec-tively (Fig. 5A). Introns 1 and 2 are large (2247 bp andTable 1: pha-4 and daf-16 are required for dauer recoveryGenotype RNAi Dauer recovery (%) a n b ppha-4 is required for dauer recoverydaf-7(e1372) control 97.9 ± 2.1 71 /daf-7(e1372) pha-4 32.1 ± 4.4 76 0.0002 cdaf-1(m40) control 99.3 ± 0.7 89 /daf-1(m40) pha-4 27.4 ± 6.0 89 0.0003 cdaf-2(e1370) control 96.0 ± 1.4 108 /daf-2(e1370) pha-4 10.4 ± 1.9 87 <0.0001 cage-1(m865) control 96.6 ± 1.9 86 /age-1(m865) pha-4 2.3 ± 1.2 84 <0.0001 cdaf-16 is required for dauer recoverydaf-7(e1372) / 97.3 ± 1.3 78 /daf-16(mgDf47); daf-7(e1372) / 41.0 ± 3.6 92 0.0001 dPHA-4 and DAF-16 function in parallel during dauer recoverydaf-7(e1372) control 97.3 ± 1.5 111 /daf-7(e1372) pha-4 44.0 ± 2.4 107 <0.0001 cdaf-16(mgDf47); daf-7(e1372) control 53.5 ± 6.2 109 0.0023 edaf-16(mgDf47); daf-7(e1372) pha-4 9.1 ± 2.1 101 0.0024 c0.0004 fa Mean percentages of dauer recovery ± standard errors.b Number of animals scored.c Daf-c mutants treated with control RNAi vs. pha-4 RNAi; p values for t-tests were calculated using the GraphPad Prism 4 software..d daf-7 vs. daf-16; daf-7 treated with OP50 E. coli food.e daf-7 vs. daf-16, daf-7 treated with control RNAi.f daf-7 vs. daf-16; daf-7 with pha-4 RNAi.pha-4 is expressed in the somatic gonadFigure 3pha-4 is expressed in the somatic gonad. (A) pha-4::gfp expression in the distal tip cells and in the intestine of a daf-2(e1370) dauer larva. (B) pha-4::gfp expression in the uterus and spermathecae of a daf-2(e1370) L4 larva. Anterior is left and dorsal is up. dtc, distal tip cell; sp, spermatheca; ut, uterus; int, intestine. Scale bars, 10 μm.Page 4 of 10(page number not for citation purposes)a Daf-d mutant, we used a daf-16(mgDf47); daf-7(e1372)strain to examine dauer recovery and adults recovered1329 bp, respectively), considering that the median sizeof confirmed introns in the C. elegans genome is 65 bpBMC Developmental Biology 2008, 8:26 http://www.biomedcentral.com/1471-213X/8/26[35]. These introns may contain promoters for pha-4b andpha-4c, or may carry cis-regulatory elements. The secondintron contains a potential DAF-16 binding site localizednear the 5' -end of the pha-4c coding region (Fig. 5A). Thissite and flanking sequences are conserved in the corre-sponding intron of the pha-4 homolog in C. briggsae (Fig.5B).We mutated the element in the pha-4::gfp construct, butdid not observe altered gfp expression in transgenic ani-mals (data not shown). It is possible that pha-4a expres-sion is not affected by the mutation. To test whether pha-4b and pha-4c are regulated through this binding site, wecloned a 3.8-kb DNA fragment of pha-4 (which carries thefirst two introns, the second exon and part of the thirdthese two constructs into daf-2 animals. Animals carryingthe construct with the normal DAF-16 binding siteshowed gfp expression only in head neurons (Fig. 5C);whereas animals carrying the mutated DAF-16 bindingsite showed very weak gfp expression in the same cells(Fig. 5D). This suggests that the potential DAF-16 bindingsite in the second intron of pha-4 has regulatory activity invivo.DAF-16 is activated and localized to the nucleus shortlyafter food deprivation [36]. We compared pha-4 mRNAlevels between well-fed and starved L3 larvae, the alterna-tive third stage to dauer larvae. Starved wild-type L3 lar-vae, which have high DAF-16 activity, had higher levels ofpha-4 transcripts than well-fed wild-type L3 larvae, whichhave low DAF-16 activity (Fig. 5E). However, starvationalso increases pha-4 transcription in the daf-16(mgDf47)null mutant background, although both well-fed andstarved daf-16 animals showed slightly lower pha-4 tran-scription compared to wild-type N2 animals (Fig. 5E).These results suggest that there may be more than onetranscriptional regulator promoting pha-4 transcription inresponse to starvation.To test the genetic interaction between DAF-16 and PHA-4, we treated the daf-16; daf-7 double mutant, which isDaf-c, with the control or pha-4 RNAi in dauer recoveryassays. pha-4 RNAi treatment further reduced recovery ofdaf-16; daf-7 dauer larvae (Fig. 5F, Table 1). This resultsuggests that DAF-16 and PHA-4 may function in parallelpathways to regulate dauer recovery.Knocking-down pha-4 by RNAi inhibits lag-2 transcription in vulval cellslag-2 is a candidate PHA-4 target, since pha-4 and lag-2 areboth expressed in DTCs in dauer larvae, and there are twopotential PHA-4 binding sites in the 1 kb lag-2 promoter.lag-2 encodes a ligand for Notch signaling pathways ingonad and vulva development [37,38]. LAG-2 expressionin DTCs regulates entry into mitosis versus meiosisthrough the GLP-1/Notch signaling pathway [38]. Dauerlarvae carrying an integrated lag-2::gfp reporter exhibitedlag-2 expression in IL1 neurons and DTCs (data notshown).We crossed the integrated lag-2::gfp reporter into the daf-2(e1370) mutant to test the effect of pha-4 RNAi on lag-2expression in DTCs and to assess possible post-dauergonadal defects. We found that lag-2::gfp expression inDTCs and IL1 neurons is not affected by pha-4 RNAi treat-ment, nor did lag-2 RNAi-treated animals show post-dauer gonad defects that were observed in animals treatedwith pha-4 RNAi (data not shown). GLD-1, a KH motif-DAF-16 regulates dauer recovery, gonad and vulva develop-mentFigure 4DAF-16 regulates dauer recovery, gonad and vulva development. (A) DAF-16 is involved in dauer recovery. The daf-16(mgDf47); daf-7(e1372) double mutant has signifi-cantly decreased dauer recovery percentage compared to daf-7(1372). Percentages of dauer recovery, numbers of ani-mals scored, and p values for t-tests are shown in Table 1. The entire experiment was performed twice with triplicates for each treatment. (B-C) DAF-16 is involved in post-dauer gonad and vulva development. (B) A daf-7(e1372) post-dauer adult, and (C) a daf-16(mgDf47); daf-7(e1372) post-dauer adult. Anterior is left and dorsal is up. vul, vulva; oo, oocytes; and emb, embryos. Scale bars, 10 μm.Page 5 of 10(page number not for citation purposes)exon) in frame with the gfp coding sequence. We thenmutated the potential DAF-16 binding site, and injectedcontaining RNA binding protein, functions downstreamof LAG-2 in the GLP-1/Notch signaling pathway in germ-BMC Developmental Biology 2008, 8:26 http://www.biomedcentral.com/1471-213X/8/26Page 6 of 10(page number not for citation purposes)DAF-16 and PHA-4 function in parallel during dauer recoveryFigure 5DAF-16 and PHA-4 function in parallel during dauer recovery. (A-E) Transcriptional regulation of pha-4 expression. (A) pha-4 gene structure (adapted from [49]). (B) The potential DAF-16 binding site in the second intron of pha-4 and flanking sequences are conserved in the corresponding intron of C. briggsae. The potential DAF-16 binding site is shown in red, capital letters. (C) A daf-2(e1370) L4 larva carrying a pha-4::gfp reporter with the potential DAF-16 binding site shows gfp expression in head neurons. (D) A daf-2(e1370) L4 larva carrying a pha-4::gfp reporter with the potential DAF-16 binding site mutated shows diminished gfp expression in head neurons. Two transgenic lines were made from each construct. 20 animals were examined from each transgenic line. Over 50% of animals showed representative expression patterns as shown in this panel. Pictures were taken using the same exposure time. (E) Increased pha-4 transcription upon starvation. Semi-quantitative RT-PCR experiments were performed to assess pha-4 transcript levels in well-fed N2 L3 larvae with low DAF-16 activity (lane 1); starved N2 L3 larvae with high DAF-16 activity (lane 2); well-fed daf-16(mgDf47) L3 larvae and starved daf-16(mgDf47) L3 lar-vae with no DAF-16 activity (lanes 3 and 4). RT-PCR experiments were performed three times with consistent results using three independent RNA preparations. (F) The daf-16 mutation and pha-4 RNAi have additive effects on dauer recovery. A daf-16(mgDf47); daf-7(e1372) double mutant treated with pha-4 RNAi showed more severe dauer recovery defects compared to either the daf-16(mgDf47); daf-7(e1372) mutant control or the daf-7(e1372) mutant treated with pha-4 RNAi. Percentages of dauer recovery, numbers of animals scored, and p values for t-tests are shown in Table 1. The entire experiment was per-formed twice with triplicates for each treatment.BMC Developmental Biology 2008, 8:26 http://www.biomedcentral.com/1471-213X/8/26line development [39]. pha-4 RNAi treatment also doesnot affect GLD-1 expression in the daf-7(e1372) mutant(data not shown). Hence, PHA-4 may not functionthrough the LAG-2-GLP-1/Notch signaling in gonaddevelopment.lag-2 is also expressed in P6.p and its descendants, thecells that adopt the 1° vulval fate [37,40,41]. LAG-2 is oneof the ligands that function through LIN-12/Notch lateralsignaling to promote P5.p and P7.p to adopt 2° vulvalfate [37]. Previous studies showed that a lag-2 tempera-ture-sensitive (ts) mutant has a protruding vulva (Pvl)when it is grown at the restrictive temperature [42]. ThePvl phenotype is similar to that of pha-4 RNAi-treated ani-mals. Thus, it is possible that LAG-2 functions down-stream of PHA-4 in vulva development. Using the samelag-2::gfp transgene as shown in previous studies [40,41],we observed lag-2::gfp expression in four P6.p descendantcells in L4 larvae. pha-4 RNAi greatly decreased lag-2::gfpexpression in those P6.p descendant cells (Figure 6).Therefore, PHA-4 is required for lag-2 expression in vulvalcells in vivo. Although electrophoretic mobility shift assays(EMSAs) indicated that PHA-4 binds to the lag-2 pro-moter in vitro (data not shown), it is not clear whetherPHA-4 directly promotes lag-2 transcription in vulval pre-cursor cells. Taken together, the data show that PHA-4 isimportant for lag-2 transcription in vulva development,but does not affect lag-2 expression in distal tip cells forgermline development.DiscussionPHA-4 plays essential roles in post-dauer developmentPHA-4 is expressed in every pharyngeal cell during embry-onic development, and it directly regulates expression ofmany pharyngeal genes [23,24,27-29]. SAGE and RT-PCRdata showed that pha-4 transcription is elevated in thedauer stage. Our RNAi analysis revealed that PHA-4 playsessential roles in dauer recovery, gonad and vulva devel-opment. Thus, PHA-4 serves as a key regulator to controldifferent aspects of embryonic and larval development.pha-4 is expressed in the somatic gonad. Previous studiessuggested that there is an unknown signal from gonadalsheath cells, uterus and spermathecae precursor cells thatregulates germline development [31]. Identification ofPHA-4 targets in gonad and vulva development may helpcharacterize this unknown signal.DAF-16 and PHA-4 function in parallel pathways during dauer recoveryTranscription factors from dauer pathways are potentialregulators of pha-4 transcription since pha-4 mRNA levelsare increased in dauer larvae. We focused on DAF-16because 1) daf-16 and pha-4 have overlapping expressionPHA-4 is required for vulval transcription of lag-2Figure 6PHA-4 is required for vulval transcription of lag-2. DR2429 daf-2(e1370); qIs56(unc-119 lag-2::gfp) animals were treated with control or pha-4 RNAi after hatching at 25°C until dauer larvae formed constitutively. The dauer larvae were then transferred to fresh RNAi plates and incubated at 15°C for 2 days until animals resumed development to the late L4 stage. Two out of four P6.p descendant cells with lag-2::gfp expression are indicated by arrow heads in a control DR2429 post-dauer L4 larva (A) and a DR2429 post-dauer L4 larva with pha-4 RNAi treatment (B). The other two P6.p descendant cells show similar lag-2::gfp expression patterns. The entire experiment was performed twice with 40 animals examined for each treatment. 37 out of 40 pha-4 RNAi-treated DR2429 post-dauer L4 larvae showed obviously diminished lag-2::gfp in four P6.p descendant cells compared to control animals. Anterior is left and dorsal is up. Scale bars, 10 μm.Page 7 of 10(page number not for citation purposes)patterns, 2) daf-16 and pha-4 have similar loss-of-functionphenotypes, and 3) there is a conserved, potential DAF-16BMC Developmental Biology 2008, 8:26 http://www.biomedcentral.com/1471-213X/8/26binding site in the non-coding region of pha-4. We haveshown that this potential DAF-16 binding site has regula-tory function on pha-4 expression in head neurons. How-ever, this regulation is unlikely to be important for theRNAi phenotypes we observed since gene expression inwild-type neurons is refractory to RNAi.pha-4 shows increased transcription under starvation,which also causes increased DAF-16 activities. This obser-vation is consistent with the recent study that PHA-4 playsimportant roles in dietary restriction-mediated life spanextension and pha-4 transcription is increased upon die-tary restriction or in the eat-2 mutant, which serves as agenetic mimic of dietary restriction [43]. However, theincreased pha-4 transcription upon starvation is inde-pendent of daf-16. This does not exclude the possibilitythat DAF-16 activates pha-4 transcription, but it suggeststhere may be multiple transcription factors that regulatepha-4 expression.We employed dauer recovery assays to characterize thegenetic interaction between daf-16 and pha-4. The daf-16mutation and pha-4 RNAi have additive effects on inhibi-tion of dauer recovery. A daf-16; daf-7 dauer-constitutivemutant treated with pha-4 RNAi showed more severedauer recovery defects compared to either the daf-16; daf-7 mutant treated with the control RNAi or the daf-7mutant treated with pha-4 RNAi. Therefore, DAF-16 andPHA-4 function in parallel pathways to regulate dauerrecovery.Other potential regulators of PHA-4The regulation of pha-4 expression and activity is complex.pha-4 expression in the intestine is inhibited by the let-7micro RNA, which is essential for the transition from theL4 larval stage to adult [44]. Interestingly, let-7 also inhib-its DAF-12 expression in hypodermal seam cells [44].Since micro RNAs usually regulate translation of themRNA, increased mRNA levels do not guarantee elevatedactivity. It will be interesting to examine whether PHA-4protein levels and activities are increased in dauer larvae.There is a tract of 29 C residues in the second intron ofpha-4 (Fig. 5A). Deletions in this sequence can be fre-quently detected in the dog-1 (DEAH helicase) mutant[45], which exhibits germline and somatic deletions ingenes containing such poly G/C tracts. However, it is notyet known whether this poly C tract plays a regulatory rolein pha-4 expression. Recently, Updike and Mango [46]have reported a screen for suppressors of pha-4 loss-of-function. One of the suppressors (px63) turned out to bean allele of pha-4, with elevated expression. The pha-4(px63) allele carries a 156 bp deletion in the seconddeletion overlaps with the polyC tract, and the insertion islikely to overlap with the potential DAF-16 binding siteand flanking sequences. Updike and Mango proposedthat the DNA sequence rearrangement in pha-4(px63)either disrupts a negative regulatory element or introducesa positive element for pha-4 expression [46]. It will beinteresting to test which mutation is responsible for theelevated pha-4 expression and whether the polyC tract orthe potential DAF-16 binding site is involved.PHA-4 is required for vulval transcription of lag-2lag-2 encodes the ligand for Notch signaling pathways inthe gonad and vulva. Our results showed that PHA-4 isimportant for lag-2 transcription in vulval cells, but doesnot affect lag-2 expression in distal tip cells for germlinedevelopment. Vulval transcription of lag-2 is regulated bythe inductive EGFR/MAPK signaling pathway, and LIN-31is the transcription factor functioning downstream of thispathway in vulva development [37]. Although we foundthat PHA-4 binds an element in the lag-2 promoter invitro, we did not detect any PHA-4 expression in vulvalcells where lag-2 is expressed using the partial PHA-4::GFPtranslational fusion reporter. More work will be requiredto determine whether PHA-4 directly promotes lag-2 tran-scription, or whether it functions through components ofthe EGFR/MAPK pathway.ConclusionDAF-16 and PHA-4 play essential roles during dauerrecovery, and PHA-4 functions upstream of LIN-12/Notchsignaling during vulval development. The dauer/non-dauer decision is determined by food, population densityand temperature [5]. The essential functions of PHA-4 inboth pharyngeal and reproductive development inresponse to environmental factors suggest a link betweennutrient in-take and reproduction. Hence, the analysis ofPHA-4 functions in dauer larvae will help explain how theoverall network of transcriptional control is formed, andhow different aspects of C. elegans development are inte-grated.MethodsStrainsStrains were maintained as described by Brenner [47].Strain names and genotypes of animals used were:GR1329 daf-16(mgDf47)I, DR2279 age-1(m875)II,CB1370 daf-2(e1370)III, CB1372 daf-7(e1372)III, DR40daf-1(m40)IV, DR2427 daf-16(mgDf47)I; daf-7(e1372)III,DR2429 daf-2(e1370)III; qIs56(unc-119; lag-2::gfp)(IV orV), DR2454 daf-2(e1370III) mEx167 [rol-6(su1006) pha-4pE3::gfp], DR2455 daf-2(e1370III) mEx168 [rol-6(su1006) pha-4pE3::gfp], DR2458 daf-2(e1370III)mEx169 [rol-6(su1006) pha-4::gfp], DR2459 daf-Page 8 of 10(page number not for citation purposes)intron, and an insertion of ≥ 5.3 kb between the 3' end ofintron 2 and the middle of exon 5 [46]. Interestingly the2(e1370III) mEx170 [rol-6(su1006) pha-4::gfp], DR2460daf-2(e1370III) mEx171 [rol-6(su1006) pha-4::gfp(M)],BMC Developmental Biology 2008, 8:26 http://www.biomedcentral.com/1471-213X/8/26and DR2461 daf-2(e1370III) mEx172 [rol-6(su1006) pha-4::gfp(M)].RNAiA 1.3-kb coding region of the pha-4 gene was amplifiedfrom C. elegans genomic DNA using primers 5' CGG AATTCG TTT TAC CAC TGG CAC CAC 3' and 5' CCC AAGCTT CTG GTA TAC TCC GTT GGT G 3'. The PCR productswere cloned into the RNAi vector L4440 between the EcoRI and Hind III sites. The pha-4 RNAi construct was trans-formed into E. coli HT115(DE3). RNAi bacteria cultiva-tion and double-stranded RNA induction were performedas described by Kamath et al. [32]. In all RNAi assays, E.coli HT115(DE3) carrying the empty RNAi vector L4440was fed to the same strain as controls.Dauer recovery assaysAbout ten Daf-c gravid adults were transferred to RNAiplates, and allowed to lay eggs for 4 to 6 hours before theywere removed. The plates were incubated at 25°C forthree days until dauer larvae formed constitutively. Thedauer larvae were then transferred to fresh RNAi platesand incubated at 15°C to resume development. Animalsthat became L4 larvae or young adults after 3 days at 15°Cwere scored as recovered. Gonad and vulva morphologiesof these adult animals were examined using a Zeiss Axio-scope with Nomarski optics.RT-PCRTotal RNA was extracted using the Trizol reagent (Invitro-gen) following the manufacturer's instructions. Reversetranscription followed by PCR reactions were performedto determine gene transcription levels from different sam-ples as previously described [48]. The following gene-spe-cific primers were used to amplify transcripts of interest:pha-4, 5' GCG GAG CTC ATG AAC GCT CAG GAC TATCTG 3' and 5' CGC AAG CTT TAG GTT GGC GGC CGAGTT C 3' ; rpl-21, 5' ATG ACT AAC TCC AAG GGT C 3' and5' TCA CGC AAC AAT CTC GAA AC 3'pha-4::gfp reportersA 5.5-kb DNA fragment that contains the 1.5-kb promoterthrough part of the third exon of pha-4 was amplifiedfrom C. elegans genomic DNA using primers 5' CAA CGAGAG GGC ATG CTG TGA AC 3' and 5' CGG GAT CCTGAT ATG GTT GGT AGT TTA ACG 3'. The PCR productswere cloned into the vector pPD95.67 (a gift from Dr.Andrew Fire) between the Sph I and BamH I sites. The pha-4::gfp clone (50 ng/μl) was injected into the ovaries of daf-2(e1370) young adults along with the pRF4 rol-6(su1006)dominant transformation marker (100 ng/μl).To test whether the potential DAF-16 binding site in theusing primers 5' ACA TGC ATG CGT AAG GCA CCA GTTATT TTC TG 3' and 5' CGG GAT CCG GCC TGC AAG AAAAAA ATT GAA AG 3'. The PCR products were cloned intothe vector pPD95.67 between the Sph I and BamH I sites.The potential DAF-16 binding site (TATTTAC) wasmutated to GCGGGCA using the QuickChange site-directed mutagenesis kit (Stratagene). The constructs (50ng/μl) with normal or mutant binding sites were co-injected into the ovaries of daf-2(e1370) young adultsalong with the pRF4 (100 ng/μl) marker.NoteIt has been reported that PHA-4 function is required forexit from the dauer stage and for development of the vulva[28,46].AbbreviationsSAGE, serial analysis of gene expression; RT-PCR, reversetranscriptase polymerase chain reaction; Daf, dauer for-mation; RNAi, double stranded RNAi-mediated interfer-ence; GFP, green fluorescent protein; DTC, distal tip cell.Authors' contributionsDC and DLR. designed the experiments and drafted themanuscript. DC carried out the experiments.AcknowledgementsWe thank P. Albert for help with Figures; S. Jones and S. Holt for help with SAGE data; G. Ruvkun for the daf-16(mgDf47) strain; J. Kimble for lag-2::gfp and lag-2(q420) strains; T. Schdel for the gld-1::gfp strain; S. Mango for shar-ing unpublished results; A. Fire for the gfp vector; and members of the Rid-dle laboratory for helpful discussion. Some strains were obtained from Caenorhabditis Genetics Center, funded by the NIH, National Center for Research Resources. This work was supported by NIH grants AG12689 and GM60151 to D.L.R. D.C. was supported by a Life Sciences Fellowship from the University of Missouri.References1. Cassada RC, Russell RL: The dauerlarva, a post-embryonicdevelopmental variant of the nematode Caenorhabditis ele-gans.  Dev Biol 1975, 46(2):326-342.2. Golden JW, Riddle DL: A pheromone-induced developmentalswitch in Caenorhabditis elegans: Temperature-sensitivemutants reveal a wild-type temperature-dependent process.Proc Natl Acad Sci U S A 1984, 81(3):819-823.3. 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