To achieve this, we systematically screened ALS/FTLD-associated and synthetic TDP-43 isoforms via same-site gene insertion and neural expression in Drosophila; followed by transposon-based motor neuron-specific transgenesis in a chick vertebrate system. Using this bi-systemic approach we uncovered a requirement of inherent TDP-43 RNA-binding function—but not ALS/FTLD-linked mutation, mislocalization, or truncation—for TDP-43-mediated neurotoxicity in vivo.Alteration and/or mutations of the ribonucleoprotein TDP-43 have been firmly linked to human neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). The relative impacts of TDP-43 alteration, mutation, or inherent protein function on neural integrity, however, remain less clear—a situation confounded by conflicting reports based on transient and/or random-insertion transgenic expression. We therefore performed a stringent comparative investigation of impacts of these TDP-43 modifications on neural integrity  This is further confounded by recent studies separately reporting that either overexpression of wild type TDP-43 (TDP-43WT), or ALS/FTLD-linked TDP-43MS, or inactivating endogenous TDP-43 in vivo in different invertebrate and vertebrate models WT and human ALS/FTLD-linked TDP-43 variant TDP-43A315T phenocopied pathological hallmarks of TDP-43-linked ALS in vivo.TAR-DNA binding protein-43 (TDP-43) is a multifunctional heterogeneous ribonucleoprotein implicated in mRNA processing and stabilization in vivo—to alleviate seemingly conflicting conclusions drawn from separate experiments addressing different TDP-43 variants in diverse systems at varying expression levels. To achieve this, we generated expression constructs for a series of TDP-43 variants, including TDP-43WT, synthetic mutants (TDP-43SM) and ALS/FTLD-linked TDP-43MS (CTF) found in cytosolic aggregates of ALS/FTLD patients ΔNLS). TDP-43 is known to effectively bind RNA 147L/F149L) into the first RNA recognition motif (RRM1) to abolish inherent TDP-43 RNA-binding function WT, TDP-43FFLL displayed a complete loss to bind to UG12 RNA oligomers in pull-down assays  analysis  controls and TDP-43WT expressing flies, indicating that TDP-43 expression had no detectable impact on endogenous TBPH transcript levels in these flies. Next we performed qRT-PCR of all TDP-43 variants normalized to endogenous actin5C and TBPH. This analysis revealed significantly higher transcript levels for TDP-43CTF. Although TDP-43A315T and TDP-43N390D expression levels were found to be slightly, but significant elevated compared to that of TDP-43WT, the overall expression levels of TDP-43MS variants were similar  cells , non-neula cells , as well neurons . The dif neurons ; Fig. S1 nucleus , while Tlization , 3B, S1B-43A315T , and all nucleus –H. TDP-4ear foci . Similar in vivo .CTF and TDP-43FFLL, ubiquitous expression of all TDP-43 variants tested resulted in premature lethality  through random insertion transgenesis. In flies expressing these variants, the intracellular localization of TDP-43 mirrored that observed for the corresponding untagged TDP-43 variants . Moreover, we identified one TDP-43WT:GFP line to cause lethality after pan-neural expression. However, this line (TDP-43WT:GFP#14) did not display a lethal phenotype after muscle or glia-specific expression  expression of TDP-43WT:GFP, TDP-43ΔNLS:GFP and TDP-43CTF:GFP on adult viability (WT:GFP (10/11 independent lines) and TDP-43ΔNLS:GFP (15/16 independent lines). Ubiquitous expression of TDP-43CTF:GFP however, was never observed to cause lethality (0/11 independent lines). Even lines with markedly higher TDP-43CTF:GFP expression levels than TDP-43WT:GFP did not result in a comparable decline in adult viability, suggesting that TDP-43CTF:GFP, similar to untagged TDP-43CTF, does not exert robust toxicity . This was based on tol2-transposon-mediated stable genomic insertion Hb9::Cre driver construct. This system facilitated stable N-terminally V5 peptide-tagged TDP-43 protein expression in late-stage chick motor neurons  or RNA-binding deficient TDP-43FFLL, in contrast, did not trigger enhanced motor neuron loss compared to control vector transfection . We therefore tested the impact on exogenous TDP-43-triggered motor neuron loss upon removing the C-terminal portion of TDP-43. To achieve this we introduced into chick motor neurons a synthetic TDP-43 variant—TDP-43NTF: TDP-43 “N-terminal fragment”—and tested for both, induction of early apoptosis and eventual effects on motor neuron numbers  to drastic . This further highlights the importance of stringently controlling relative protein levels when investigating the activity of different TDP-43 isoforms. Accordingly, less overt variant-specific differences in toxicity could consequently be masked, or misrepresented, by varying TDP-43 concentrations—such as achieved by random-insertion transgenesis. For instance, low TDP-43WT expression levels had only mild impacts on longevity, while high levels of TDP-43WT resulted in a marked reduction of life-span , work dealing with Drosophila stock center. The site-directed and random-inserted UAS:TDP-43 transgenic lines were generated by germline transmission (BestGene). Strain PBac{yellow[+]-attP-3B}VK00002  was used to generate the site-directed insertions of different TDP-43 variants.All fly stocks were maintained and raised on standard cornmeal-yeast-agar medium. Unless otherwise noted, all fly strains used in this study are available at the Bloomington Amino acid changes of TDP-43[WT] were introduced by site-directed mutagenesis using following primers (sequence 5′-3′):TCAAAGGGGTTAGGCTTAGTTCGTTTTACGGF147L/F149L.For: CCGTAAAACGAACTAAGCCTAACCCCTTTGAF147L/F149L.Rev: GCTTTGGGAATCAGAGTGGATTTGGTAATAG287S.For: TATTACCAAATCCACTCTGATTCCCAAAGCG287S.Rev: GGGATGAACTTTGGTACGTTCAGCATTAATA315T.For: ATTAATGCTGAACGTACCAAAGTTCATCCCA315T.Rev: CAGAACCAGTCATGCCCATCGGGTAG348C.For: TACCCGATGGGCATGACTGGTTCTGG348C.Rev: CTAATTCTGGTGCAACAATTGGTTGGGGAA382T.For: TCCCCAACCAATTGTTGCACCAGAATTAGA382T.Rev: GATCAGCATCCGATGCAGGGTCGN390D.For: CGACCCTGCATCGGATGCTGATCN390D.Rev: in vitro mutagenesis was performed using QuikChange® Site-Directed Mutagenesis Kit (Stratagene) according to manufacturer's manual.pENTR1A-TDP-43 was used as template. BamHI and 3′-XbaI restriction sites, respectively (see below). Subsequent digest with respective enzymes allowed sub-cloning into pUASattB vector (cut with BglII and XbaI).For generation of transgenic flies, TDP-43 variants were amplified using primers introducing 5′-TTCAGTCGACTGGATCCTATAGGGAGpENTR1A.For: GTCTAGACTACATTCCCCAGCCAGAApENTR1A.Rev: BamHI/HindIII) into pcDNA3.1(-) (Invitrogen) with an inserted 5′-Flag tag (NotI/EcoRI).For expression in human cells, respective TDP43 variants were subcloned in frame , MmCherry open reading frame and a terminating SV40 poly-A signal, and was inserted between the 2Tol-sites as a 5′SspI/3′XhoI fragment. The 2Tol vector IRES as a 5′XbaI/3′SmaI fragment after PCR amplification using the following primers:The XbaI.For: GCTCTAGAATGTCTGAATATATTCGGGTAACCTDP-43 SmaI.Rev: TCCCCCGGGCTACATTCCCCAGCCAGAAGACTTTDP-43 CTFXbaI.For: GCTCTAGACTGCGGGAGTTCTTCTTDP-43NTFSmaI.Rev: CCCGGGGGACTATTAAGCATCTGTCTCATCCATTTTDP-43st chromosomal P{w[+mW.hs] = GawB}elav[C155]  was used to drive the different TDP-43 variants. For embryonic-onset longevity analysis, C155::Gal4elav virgins were crossed to males carrying different TDP-43 variants . The F1 generation was raised at 25°C, male flies with driver and respective TDP-43 transgenes  were used for analysis. Driver flies  served as control. Statistical analysis was performed using GraphPad Prism.For pan-neural expression, 1c155::Gal4;Gal80[ts]elav) were crossed to w/Y;UAS::TDP-43ICyO males, respectively. The F1 generation was raised at 18°C to abolish TDP-43 expression and male flies C155::Gal4/Y;UAS::TDP-43IGal80[ts]elav were analyzed. In both paradigms, groups of 10-20 male flies (0-48 hours post hatching) per vial were shifted to 29°C, and dead flies were counted daily. Fresh food vials were provided every second day. At least 60 flies were analyzed per genotype.In case of adult-onset expression, virgins carrying a temperature sensitive Gal80 in combination with the pan-neural driver P{w[+mW.hs] = GawB}elav[C155]; P{w[+mC] = tubP-GAL80[ts]}20  was used to drive motor neuron-specific expression of TDP-43 variants. Flies were raised at 25°C, male flies  were collected (0–48 hours post hatching) and shifted to 29°C. Individual flies were tested 3 times per time point  for negative geotaxis (ability to climb 8 cm in 10 s). At least a total number of 60 flies was used per time point and genotype. Statistical analysis was performed using GraphPad Prism.The 3Heads of flies were dissected and homogenized in RIPA buffer , 1% Nonidet P-40). Lysates were separated on 12% Gel by SDS-PAGE. Proteins were transferred to nitrocellulose membrane. Membrane was blocked with 5% (w/v) skimmed milk Tris buffered saline containing 0.1% Tween 20. For detection of proteins the following primary antibodies were used: anti-TARDBP , anti-Syntaxin ). Secondary HRP-coupled antibodies  were used for chemiluminiscence detection .Heads of flies with pan-neural expression of different TDP-43 variants were isolated using Trizol (Invitrogen). For qRT–PCR, 1 µg total RNA was reverse transcribed using Transcriptor High Fidelity cDNA Synthesis kit (Roche) and anchored oligo-dT primer. 1/10 dilutions were used in triplicates with 0.2 µM primer and 5 µl LightCycler 480 SYBR Green I Master in a 10 µl reaction and qPCR executed in a 384-well block on a LightCycler 480 system (Roche). Amplification efficiencies of targets and references were determined and used for further calculations of normalized relative (Delta DeltaC(T)) expression levels according to CACACCGTGCCCATCTACGAGGActin-5C.For: CTTCTGCATACGGTCGGCGATGCActin-5C.Rev: ACTGAGGATGAGCTGCGGGAGTTCTDP-43.For: CAAAGAGACTGCGCAATCTGATCATCTGTDP-43.Rev: GGAAGGGGCCGAATAACCCGAACTBPH.For: CACACATCATTGGGTGACAGGCACCTBPH.Rev: 2/air. DNA transfections were performed with FuGene6 (Roche) according to manufacturer's instruction.HEK293E cells (Invitrogen) were grown in Dulbecco's modified Eagle medium (DMEM), supplemented with 10% fetal bovine serum (PAA) at 37°C under humidified 5% COin vitro transcribed biotinylated RNA to protein lysate was performed as described recently TAATACGACTCACTATAGGG) were diluted in 47 µl annealing buffer (30 mM Hepes (pH 7.4), 100 mM KAc, 2 mM MgAc) and heated to 95°C for 5 min. After cooling down slowly, 12 µl of annealed oligos were in vitro transcribed/biotinylated using Biotin RNA Labeling Mix and T7 RNA Polymerase (both Roche) according to manufacturer's instructions. After digest of DNA, 2 µl of this reaction were incubated with 250 µl RNA binding buffer (20 mM HEPES (pH 7.5), 5 mM MgCl2, 50 mM KCl, 150 mM NaCl, 0.5 mM EGTA, 0.5 mM dithiothreitol, 10% glycerol) and 1,000 µg HEK293 lysate for 20 min at 30°C. HEK293 lysates were generated from HEK293 cells transfected with Flag-TDP-43 wild type or F147L/F149L for 48 h and lysed in RNA binding buffer +1% Triton X-100. After UV irradiation and brief RNase A digest, Streptavidin-beads (Sigma) were added and precipitations carried out overnight at 4°C. Eluate and input were separated by 10% SDS-PAGE, blotted onto nitrocellulose and probed overnight with antibodies against TDP-43 , Flag  and GAPDH  as a loading control.UV-crosslinking of Gallus gallus) eggs were obtained from Horstmann Geflügelzucht GmbH. Eggs were incubated at 37.5°C and 80% humidity (J. Hemel-Brutgeräte GmbH) for 72 hours (∼3 days) to obtain embryos at embryonic day 3 (E3). Microinjections were performed using a micropipette needle made from pulled glass capillary tubes. The needle was loaded with a mixture of DNA, Hb9::Cre (1.0 µg/µl) for motor neuron-specific expression, pCAGGS::T2TP (1.0 µg/µl) and LTEV containing the TDP-43 variant (concentration of 1.0 µg/µl). Up to 5 ml of egg white was removed using a syringe. Afterwards the egg was windowed in the middle. Egg yolk and the embryo stayed unharmed during this procedure. The DNA-mixture got injected into the neural tube (lumbar region) of the embryo. The injection site was electroporated using an ECM 830 electroporation system . The electrodes were placed in parallel so that the developing spinal cord was situated between the electrodes. Electroporation with the electrodes placed in this way transported DNA located in the spinal cord towards the positive electrode, resulting in approximately half of the spinal cord being transfected. The electroporation settings were 5 pulses of 25 mV for 50 ms in the LV 99 ms/500 V modus. After electroporation, the window on the operated egg was sealed with clear sellotape, and the egg was returned to the incubator.Fertilized chick , while E5 spinal cords were left intact in the embryo to minimize damage. Tissues were fixed by immersion in 4% PFA (in 1x PBS)  and then washed in 1x PBS overnight. Afterwards the tissue was incubated in 30% sucrose (in 1x PBS) overnight. All previous steps were taken out at 4°C. Ready fixed tissue was imbedded into OCT . Transversal cryosections of the spinal cord were cut in a CM1900 cryostat  at −20°C and placed on a super frost glass slide.6 w/v) at RT for 10–20 min. After DAPI staining, the slides were washed with 1x PBS, mounted with 50% glycerol and cover slipped. Primary antibodies: rabbit anti-Isl1/2 , mouse anti-Isl1 , mouse anti-V5 . Secondary antibodies: Alexa 647 rabbit anti-mouse , Alexa 488 mouse anti-rabbit , Alexa 647 mouse anti-rabbit , Alexa 488 rabbit anti-mouse . All images of mounted slices were collected using a Leica TCS/MP confocal/two-photon microscope. All images were acquired from 30 µm sections of the chick embryo spinal cord at the developmental stages of E5 and E9. Images of the E9 sections were acquired by using the 20x objective with 2x zoom. Images of the E5 sections were acquired with the 40x objective and 2x zoom. For both developmental stages  all images were a total of a 50–80 µm stack of 10 z-sections subsequently collapsed to a 2D rendering using ImageJ. Motor neuron survival was measured by counting Isl1/2 positive cells in the ventral horn, electroporated vs. non-electroporated side. The counting was performed using the Olympus Cell∧M imaging station.Chick immunofluorescent staining was performed on 30 µm slices of prefixed E5 and E9 chick embryo spinal cord. For primary antibody detection the slides were incubated in a humidified box overnight at 4°C, utilizing 1% BSA (fraction V) in 1x PBS with 0.5% Triton X-100 to enhance tissue penetration. Slides were washed with 1x PBS before the secondary staining, which was carried out with the same detergent for 30 min–1 h at RT. Slides were washed again with 1x PBS and stained with DAPI  and fixed in PBS +4% PFA for 20 min at RT. Primary antibody detection was performed at 4°C in PBST supplemented with 5% BSA (fraction V) over night. Tissue was washed in PBST before secondary antibodies were applied (2 h at RT). Specimen was mounted in Vectashield® mounting medium (Vector Laboratories) +/− DAPI. Primary antibodies: mouse anti-TDP-43 . Secondary antibodies: Alexa 488 goat-anti mouse . Alexa Fluor 568 phalloidin (Invitrogen) was used to stain F-actin (1 unit/staining) and for confocal analysis, DNA was visualized using Sytox® Orange . Epifluorescent pictures were acquired at Olympus BX51 microscope (10x or 40x objectives) using Cell∧F software. Confocal sectioning was performed at Leica TCS/MP confocal/two-photon microscope using 20x objective with 2x zoom.HEK293E cells were plated onto glass coverslips coated with PDL (Sigma) and collagen (Cohesion), fixed with 4% (w/v) paraformaldehyde and permeabilized with 1% Triton X-100 48h after transfection with Flag-tagged TDP-43 variants. Cells were incubated with primary antibodies  followed incubation with secondary antibodies anti-mouse IgG Alexa Fluor-and anti-rabbit Alexa Fluor-568 (Molecular Probes) diluted 1:2000. Nuclei were stained with Hoechst 33342 (Molecular Probes) diluted 1:5000. Coverslips were mounted onto microscope slides using fluorescent mounting medium (Dako). Confocal fluorescent images were taken with an AxioImager microscope equipped with an ApoTome Imaging System (Zeiss).Figure S1Drosophila. Salivary glands of Drosophila larvae were used to address subcellular localization of TDP-43 variants due to their large cell and nucleus size. Epifluorescence pictures of gland cells stained for TDP-43 (green) and f-Actin (red) using phalloidine (upper panel). Nuclear localization of TDP-43WT and TDP-43MS is visualized by colocalization with DAPI (blue) stained nuclei (middle panel) and illustrated by plotted fluorescence distribution (lower panel). Line indicates distance used for fluorescence measure. Note that of the assayed TDP-43 variants, only TDP-43ΔNLS displayed robust cytoplasmic localization (B).Localization of TDP-43 in non-neuronal cells of (4.10 MB TIF)Click here for additional data file.Figure S2Drosophila. We were not able to efficiently detect TDP-43CTF with our TDP-43 antibody directed to the N-terminal portion of the protein. To circumvent this problem, in addition to φ-C31 site-specific recombination, we also utilized random-insertion transgenesis in Drosophila to generate C-terminal GFP-tagged TDP-43 variants for TDP-43WT (A), TDP-43ΔNLS (B) and TDP-43CTF (C), . This allowed us to visualize TDP-43:GFP (green) directly. First we tested the different variants in non-neuronal cells of the salivary glands of L3 larvae (left). Epifluorescence analysis revealed that GFP-tagged TDP-43WT and TDP-43ΔNLS localized alike the untagged variants. (A) TDP-43WT:GFP showed nuclear localization, evident by colocalization with DAPI stained DNA (blue). (B) TDP-43ΔNLS:GFP was found to evenly distributed in the cytoplasm and almost not detected in the nucleus. Thus, the C-terminal GFP-tag did not alter localization, as it was also observed in HEK cells (not shown). (C) TDP-43CTF:GFP showed a strong cytoplasmic as well as a nuclear localization, similar to the situation observed in chick . Expression of all three constructs in photoreceptors  resulted in a robust accumulation of GFP signal in targeted cells of the eye disc (not shown). As expected, larvae expressing TDP-43WT:GFP did not display any GFP signal in photoreceptor projections (A), as localization was restricted to the nuclei of the eye disc . This indicates that cytoplasmic ΔNLS and CTF variants of TDP-43:GFP were distributed throughout neurons and were not restricted to the soma. Scale bar indicates 100 µm.Subcellular localization of TDP-43:GFP variants in see also . In cont(2.36 MB TIF)Click here for additional data file.Figure S3Gallus. Subcellular localization of TDP-43 variants (red) in E9 chick motor neurons (large DAPI+ nuclei: white). In analogy to cytosolic and nuclear localization observed in flies, TDP-43CTF (A) in chick localized alike and displayed frequent cytosolic foci (arrowheads). To test if the lack of RNA-binding also abolishes toxicity of cytoplasmic TDP-43, we combined RNA-binding deficient FFLL with the mutated NLS and generated TDP-43ΔNLS,FFLL. As observed for TDP-43ΔNLS . Thus, interference with RNA-binding of TDP-43 did not alter localization. Interestingly, TDP-43ΔNLS,FFLL was found to induce significantly lower Caspase-3 activation as compared to TDP-43ΔNLS  might display marked effects after expression in chick motor neurons. Although we achieved high expression levels and nuclear localization of TDP-43NTF in motor neurons (C), we could not detect elevated motor neuron loss Click here for additional data file.Figure S4WT:GFP lines displaying different expression levels in Western blot analysis were assayed for longevity after pan- neural expression. The line with stronger expression, TDP-43WT:GFP#16 displayed an earlier lethality as compared to the weaker expressing line TDP-43WT:GFP#10. This indicates that toxicity induced by pan-neural expression of TDP-43WT is dose-dependent. According to the GFP-Tag, we were able to compare protein abundance of different TDP-43CTF:GFP transgenes. Compared to TDP-43WT:GFP#10, TDP-43CTF:GFP#14 showed higher levels of protein expression. In spite of these high protein levels, longevity was not as strongly reduced as observed for TDP-43WT:GFP expressing flies. (B) Verification of pan-neural adult-onset expression in Western blot analysis. Flies were raised at restrictive condition , and TDP-43 expression was induced 2 days post eclosion by a temperature shift (29°C). Head lysates taken at indicated time points were analyzed for TDP-43 expression. No TDP-43WT:GFP expression could be detected under rc, but was induced at 29°C. Increasing TDP-43 protein levels were detected over time. (C) Flies with adult-onset pan-neural expression of indicated TDP-43 variants were assayed for longevity. Note that line TDP-43WT:GFP#14, lethal in embryonic-onset expression paradigm, survived to adulthood in the adult-onset expression, but displayed a very short longevity after induction of expression. For untagged and site-directed TDP-43 transgenes, the order of toxicity is comparable to the embryonic-onset paradigm (see Reduction in longevity is dose-dependent and inducible in an adult-onset expression system. (A) Two TDP-43digm see .(0.55 MB TIF)Click here for additional data file.Video S1WT expressing flies. Example of a fly with pan-neural expression of TDP-43WT. Note the lateral “hanging wing” phenotype. Dependent on expression strength and time, TDP-43WT flies lost their ability to fly, had reduced escape behavior (even if pushed with a brush) and were not able to reach an upright position after being placed dorsal side down in a reasonable time.Abnormal locomotion and wing posture in aged TDP-43(1.35 MB MOV)Click here for additional data file.


Metastasis is a process by which cancer cells learn to form satellite tumors in distant organs and represents the principle cause of death of patients with solid tumors. NSCLC is the most lethal human cancer due to its high rate of metastasis.Lack of a suitable animal model has so far hampered analysis of metastatic progression. We have examined c-MYC for its ability to induce metastasis in a C-RAF-driven mouse model for non-small-cell lung cancer. c-MYC alone induced frank tumor growth only after long latency at which time secondary mutations in K-Ras or LKB1 were detected reminiscent of human NSCLC. Combination with C-RAF led to immediate acceleration of tumor growth, conversion to papillary epithelial cells and angiogenic switch induction. Moreover, addition of c-MYC was sufficient to induce macrometastasis in liver and lymph nodes with short latency associated with lineage switch events. Thus we have generated the first conditional model for metastasis of NSCLC and identified a gene, c-MYC that is able to orchestrate all steps of this process.Potential markers for detection of metastasis were identified and validated for diagnosis of human biopsies. These markers may represent targets for future therapeutic intervention as they include genes such as Gata4 that are exclusively expressed during lung development. Metastasis is a complex multistep process by which cells in the primary tumor become able to enter and leave the vasculature, settle at distant sites and eventually grow to macroscopic secondary tumors RAF transgene specifically expressed in lung alveolar type II epithelial cells RAF other transgenes that encode proteins modulating signaling pathways frequently altered in human NSCLC allowed us to unravel two key steps in the process of metastasis, induction of the angiogenic switch and progression to micrometastasis associated with reprogramming of intestinal selector genes While work with human tumor cell lines in mice has been enlightening, lack of knowledge about their repertoire of cancer genes as well as the inability to study the kinetics of spontaneous tumor progression in these models limit their usefulness. We have therefore established a mouse model for human non–small-cell lung cancer (NSCLC) in which premalignant adenoma is induced by a alveolar papillary/columnar epithelial cells (APECs) that are the most rapidly growing tumor cells and also predominate in liver metastasis. Moreover c-MYC induces production of VEGF by tumor cells leading to tumor vascularisation with unusual vessels that are mosaics containing PECAM-1 positive and PGP 9.5 positive endothelial cells. PGP 9.5 was previously known as a marker of immature neuroendocrine cells As 50% of human NSCLC carry mutations in genes that encode activators of the mitogenic cascade (RAS-RAF-MEK-ERK) To test whether C-RAF and c-MYC cooperate in transformation of lung epithelial type II cells in vivo, SpC-C-RAF BxB mice were crossed with SpC-c-MYC transgenic mice. Observation for a period of ≥2 years demonstrated accelerated death in compound (SpC-C-RAF BxB/SpC-c-MYC) mice  althoughmetastasis initiating cells (MICs)  . Intrigus (MICs) . As expes (MICs) . Pleomors (MICs)  which als (MICs) . Of the s (MICs)  [23]. c-s (MICs) . In facts (MICs) . Prolongs (MICs) .We next looked at Aquaporin 5 expression that is shared between type I and type II pneumocytes In contrast to lungs of SpC-c-MYC single transgenic mice that only develop premalignant lesions, tumor nodules in lungs of compound or late SpC-c-MYC single transgenic mice were rapidly expanding and frequently contained macroscopic tumors with columnar cells that were absent in SpC-C-RAF BxB–driven adenomas . SeveralPapillary tumors grow faster than cuboidal tumors of both genotypes. Consistently the fraction of PCNA positive, pro SP-C expressing cells was higher in papillary tumors . SimilarIn compound mice tumor acceleration was evident histologically at all ages analyzed with frequent noninvasive macroscopic lung tumors which were absent in SpC-C-RAF BxB single transgenic mice .c-MYC has been reported to induce angiogenesis A striking observation was made when we explored potential neuroendocrine features of our NSCLC by staining with PGP 9.5. PGP 9.5 is an immature neuroendocrine marker and a candidate tumor marker for human NSCLC where it is expressed in tumor cells from >70% of stage II and IIIA tumors The angiogenic switch was presumably not accompanied by typical epithelial mesenchymal transition (EMT) at the tumor edges as we did not detect cells doubly positive for pro SP-C and vimentin or pro SP-C and N-cadherin . MoreoveK-Ras and LKB1 genes   was injencidence  and ratencidence  was incrncidence . Analysincidence . The comK-Ras and LKB1 mutations in lung tumors primarily of SpC-c-MYC single transgenic mice suggested that we might be able to use mutation data for lineage tracing. We therefore examined the DNA of liver and lymph node metastases for the mutations present in the primary tumors. Surprisingly in case of K-Ras mutations, four out of the five cases of c-MYC tumor derived metastases that could be examined in this way were negative for the mutation as was the one case of a compound mouse that had a K-Ras exon 1 mutation in the primary tumor and metastasis to lymph nodes  and maintained as heterozygotes. Compound mice were obtained by intercrossing heterozygotes. Other details of transgenic mice are described in Supporting Information .5′- TTC CCC TAC CCT CTC AAC GAC AG – 3′and human MYC anti-sense: 5′- TCC TTA CTT TTC CTT ACG CAC AA- 3′were used for amplification of human c-MYC. Actin was used for loading control.Semi quantitative RT-PCR was performed on total lung RNAs extracted from 6 weeks old mice using TRIZOL (Invitrogen) reagent as previously described (Bioluminescence imaging of luciferase expression in compound mice (SpC-C-RAF BxB/SpC-rtTA/tet-O-c-MYC) was performed as described Animals were sacrificed and organs were fixed with 4% PBS buffered formalin. Histology was done on formalin-fixed, paraffin-embedded lung specimen. 6 µm-cut sections were deparaffinized, rehydrated in graded alcohols and haematoxylin-eosin stained. Details of staining protocols are given in Supporting Information .Preparations of embedded tissues and quantitative assays for lung tumor development are described in Supporting Information .t test (two-tailed) was used to compare two groups (P<0.05 was considered significant).Tumour-free survival and comparisons of tumour numbers and tumour volume were analysed using GraphPad Prism4 software. Data are presented as mean±s.e.m. Student's Supplemental data include Supporting Information, 17 figures and one table.Text S1Supporting Material Methods and Supplementary figure legends(0.16 MB DOC)Click here for additional data file.Figure S1Premalignant lesions of SpC-c-MYC mice express type II pneumocyte- and progenitor- cell markers. Paraffin embedded lung sections were stained as indicated. Clusters of pleomorphic cells that represent premalignant lesions in SpC-c-MYC mice were highlighted with red circles. Scale bar: 50 µm.(7.93 MB TIF)Click here for additional data file.Figure S2Rescue of cryptic MYC transformants by co-expression of RAF. (A) Paraffin embedded lung sections from all genotypes were stained for active caspase 3 (brown) to detect apoptotic cells. Yellow circles identify neoplastic lesions. Jurkat cells treated or untreated with etoposide were used as positive and negative control, respectively. Haematoxylin (Blue) was used for counterstaining. Scale bar: 50 µm. (B) Quantitation of apoptotic cells for indicated genotypes. At least 40 randomly selected lesions from 3 mice per genotype were analysed for quantitation of apoptotic cells.(2.28 MB TIF)Click here for additional data file.Figure S3Conditional expression of c-MYC in lung alveolar type II cells induces tissue destruction. (A) Schematic diagram showing the generation of compound (SpC-rtTA/tet-O-c-MYC) mice conditionally expressing c-MYC in type II pneumocytes. Hs: Human. (B) Semi-quantitative RT-PCR showing inducible transgenic c-MYC (human) expression in lungs of compound mice after one week DOX administration. (C) Lung of a compound (SpC-rtTA/tet-O-c-MYC) mouse after four weeks induction shows severe tissue loss (white arrows) as evident from inspection of whole lung and the H&E stained section. Active caspase 3 staining identifies apoptotic cells (brown cells indicated by red arrows) in alveoli one day after doxycycline administration. Haematoxylin (blue) was used for counterstaining. (D) H&E staining of a lung section from four weeks-induced compound (SpC-rtTA/tet-O-c-MYC) mouse shows isolated pleomorphic cell clusters (yellow dash marking). (E) H&E staining of a lung section from 26 weeks-induced compound (SpC-rtTA/tet-O-c-MYC) mouse shows a lung adenocarcinoma with columnar cells. Scale bar: 100 µm.(3.53 MB TIF)Click here for additional data file.Figure S4Induction of phenotypic switch from cuboidal to alveolar papillary/columnar epithelial cells (APECs). (A) H&E staining of a mixed  lung tumor section from four months old compound (SpC-C-RAF BxB/SpC-c-MYC) mouse. (B) Schematic diagram showing the generation of triple transgenic compound (SpC-C-RAF BxB/SpC-rtTA/tetO-c-MYC) mice. (C) H&E staining of lung tumor sections from inducible (SpC-C-RAF BxB/SpC-rtTA/tetO-c-MYC) compound mice shows the kinetics of columnar cell appearance. D: day, W: week, M: month. Right hand panel is a magnification of the yellow box. Scale bar: 100 µm. (D) Six weeks old compound mice were imaged for in vivo luciferase expression following one week On DOX/4 weeks Off DOX schedule demonstrating inducibility. H&E staining of a lung tumor section of the On/Off DOX mouse. Inset highlights persistent papillary tumor area indicated by yellow box.(3.75 MB TIF)Click here for additional data file.Figure S5Immunostaining of emerging and late tumors of compound mice (SpC-C-RAF BxB/SpC-c-MYC) for lineage and progenitor cell markers. (A) Emerging tumors from 2 weeks-old mice were stained as indicated. (B) Late tumors from 10–16 months-old mice were stained as indicated. Markers are as indicated. In the case of fluorescence stainings colours correspond to the indicated proteins. Dapi (blue) illustrates nuclei. (C) β-catenin (green) staining of a lung tumor showing membrane localisation. Dapi (blue) illustrates nuclei.(7.15 MB TIF)Click here for additional data file.Figure S6Induction of angiogenic switch by c-MYC. (A) Immunostaining of lung tumor sections from control and metastatic animals of indicated genotypes for blood (Pecam 1) and lymph (Prox 1) vessels. Scale bar 100 µm.  Quantitation of vessel density. 5 mice per genotype were analysed. Values represent SD of mean. P values are as indicated. (D) PGP 9.5 immunostaining of lung tumor sections. Scale bar 100 µm. (E) Frozen lung sections from wild type (wt) and compound mice were stained for PGP 9.5 (red) and PECAM-1 (green) for co-expression. Dapi illustrates nuclei. Scale bar 50 µm.(5.74 MB TIF)Click here for additional data file.Figure S7VEGF induction by MYC in primary lung tumors and NSCLC cell lines. (A) Immunostaining of lung tumor sections from age-matched mice of the indicated genotypes for VEGF (brown). Haematoxylin (blue) was used for counterstaining. Scale bar: 100 µm. (B) Immunocytochemistry of human A-549 and mouse MLE-15 NSCLC cell lines for VEGF (green). Note increased VEGF expression in cells infected with Myc expressing retroviruses J5-1 and J2-11. One representative cell clone is shown for each virus. Dapi (blue) illustrates nuclei. Scale bar: 10 µm.(3.77 MB TIF)Click here for additional data file.Figure S8No evidence for EMT in tumor progression. (A) E-cadherin immunofluorescence staining (brown) of lung tumor section from a 12 months old compound mouse. Scale bar: 100 µm. (B) Paraffin embedded lung tumor sections from control and metastatic animals were stained for EMT markers as indicated. Tumor cells were marked by pro SP-C (green) staining. Dapi (blue) illustrates nuclei. Scale bar: 100 µm.(3.53 MB TIF)Click here for additional data file.Figure S9Inducible expression of c-MYC in SpC-C-RAF BxB lung tumors give rise to liver metastasis with APECs. (A) H&E staining of a lung section from a seven months induced compound mouse shows large lung tumors with columnar cell. Scale bar: 100 µm. (B) Pan-cytokeratin staining (brown) of a regional lymph node section from eleven months-induced compound mouse (SpC-C-RAF BxB/SpC-rtTA/tet-O-c-MYC) shows micrometastasis. (C) Inspection of a liver from a seven months induced compound (SpC-C-RAF BxB/SpC-rtTA/tet-O-c-MYC) mouse shows a tumor nodule in the liver (red circle). (D) H&E staining of the liver metastasis shown in C demonstrates papillary tumors with stroma (S). Scale bar: 100 µm.  Pan-cytokeratin (E) and pro SP-C immunostaining of the liver metastasis shown in C. Isolated pan-cytokeratin and pro SP-C positive tumor cells that were embedded in the stroma (S) were indicated by red arrows.(6.17 MB TIF)Click here for additional data file.Figure S10Staining of primary tumor and lung metastasis developing after subcutenous injection of A-549 J5-1 cells for chicken c-MYC.(3.35 MB TIF)Click here for additional data file.Figure S11Histopathology of metastasis to distant organs in old SpC-c-MYC mice. Multiple target organs are involved in case of a mutant K-Ras positive SpC-c-MYC lung tumor at age 20,5 months. Tissue sections were stained with indicated markers which identifies derivation of metastasis from lung adenocarcinoma. Haematoxylin (blue) was used for counterstaining.(3.71 MB TIF)Click here for additional data file.Figure S12Cystic and papillary forms coexist in late liver metastasis. (A) Macroscopic liver metastasis in an 18 months old SpC-c-MYC mouse. Note multiple solid tumor nodules  and cystic lesion (yellow arrow). (B) H&E staining of liver metastasis confirm coexistence of both papillary and cystic (yellow circle) lesions in the same organ.(2.83 MB TIF)Click here for additional data file.Figure S13Cystic liver metastasis of a 13 months old SpC-c-MYC mouse. High magnification of red inset illustrates presence of pro SP-C positive (brown) cells (arrows) in the epithelial layer lining the cyst. Haematoxylin (blue) was used for counterstaining.(1.35 MB TIF)Click here for additional data file.Figure S14Expression of transgene markers in liver metastases. Immunofluorescence staining of papillary lung tumors and liver metastasis for expression of C-RAF (green) and c-MYC (red) transgenes.(1.36 MB TIF)Click here for additional data file.Figure S15No evidence for BASC in liver metastasis. (A) Paraffin embedded sections from 18 months liver metastasis were stained for CCSP and pro SP-C. Haematoxylin was used for counterstaining. Scale bar: 50 µm. (B) The same sections were subsequently examined for co-expression of CCSP (green) and pro SP-C (red) to search for BASCs. Dapi (blue) illustrates nuclei.(3.65 MB TIF)Click here for additional data file.Figure S16Gata6/Gata4 switch in SpC-c-MYC and compound mice. (A) Reciprocal expression of Gata4 and Gata6 in primary tumor of SpC-c-MYC and compound mice. Stainings as indicated. Note decrease in the level of Gata6 expression concomitant with heterogenous Gata4 expression in late stage lung tumors. Duodenum and embryonic lung sections were used for positive control for Gata4 and Gata6, respectively. (B) Alcian Blue 2.5 expression in lung tumors from indicated genotypes. Sections from 12 months old mice were stained with Alcian Blue for mucin secretion .(6.04 MB TIF)Click here for additional data file.Figure S17Early and mutually exclusive expression of ectopic Gata4. (A) A representative lung section from a six-months-old compound (SpC-C-RAF BxB/SpC-c-MYC) mouse with an array of Gata4 expressing (brown) cells. (B) Immunohistochemistry for Gata4 and pro SP-C in serial sections of a lung tumor from a 18-monthd-old SpC-c-MYC single transgenic mouse illustrating mutually exclusive expression of both markers.(2.63 MB TIF)Click here for additional data file.


Cooperative TGF-β/EGFR signaling promotes cell migration and induces expression of both proteases and protease inhibitors that regulate stromal remodeling resulting in the acquisition of an invasive phenotype. In one physiologically relevant model of human cutaneous SCC progression, TGF-β1+EGF stimulation increases the production of several matrix metalloproteinases (MMPs), among the most prominent of which is MMP-10—an MMP known to be elevated in SCC in situ. Activation of stromal plasminogen appears to be critical in triggering downstream MMP activity. Paradoxically, PAI-1, the major physiological inhibitor of plasmin generation, is also upregulated under these conditions and is an early event in progression of incipient epidermal SCC. One testable hypothesis proposes that TGF-β1+EGF-dependent MMP-10 elevation directs focalized matrix remodeling events that promote epithelial cell plasticity and tissue invasion. Increased PAI-1 expression serves to temporally and spatially modulate plasmin-initiated pericellular proteolysis, further facilitating epithelial invasive potential. Defining the complex signaling and transcriptional mechanisms that maintain this delicate balance is critical to developing targeted therapeutics for the treatment of human cutaneous malignancies. The emergence of highly aggressive subtypes of human cutaneous squamous cell carcinoma (SCC) often reflects increased autocrine/paracrine TGF- Nonmelanoma skin cancers (NMSCs)  are the most common human malignancies , 2. In Nras gene mutation/activation/amplified expression (10–30% incidence), and inactivation of cell cycle inhibitors [ras expression may be cell type-dependent [ras gene [R172H) results in increased skin tumor formation/progression and metastatic spread [ The emergence of epithelial skin tumors appears causally linked to ultraviolet (UV) radiation exposure. Specific UV-B “signature” base changes (C→T or CC→TT), particularly in codons 177  and 278 (SCC) in the tumor suppressor p53 gene –8, likelhibitors –15. Whilependent , moleculependent , 17 are ras gene , 18. At β (TGF-β)) [β1 is a particularly robust initiator of epithelial “plasticity” , a likely facilitator of tumor invasion and metastasis ; epidermal growth factor (EGF); platelet-derived growth factor (PDGF); transforming growth factor-(TGF-β)) –24. TGF-e, e.g., , 24). Thβ1 (TGF-β1) in the tumor microenvironment appears causally linked to creation of highly aggressive metastatic variants [β1-mediated growth suppression, moreover, is frequently accompanied by mutation, allelic loss, or misregulation of elements within the TGF-β1 signaling network  . Such se, e.g., –30. The asticity , 31–37. β1-induced, EMT-associated genes in transformed human keratinocytes [ras-expressing), dual growth factor (TGF-β1+EGF)-stimulated model of EMT in transformed human keratinocytes (HaCaT II-4 cells)  has significantly enhanced our understanding of the biology of human cutaneous tumor progression while also providing a comparative map of expressed/repressed genes in actinic keratosis and SCC versus normal skin , 39. Altinocytes , 40. PAIinocytes . Use of 4 cells)  and micr4 cells) ; Table 1le actin , and a gle actin ; Table 1e, e.g., , 38–41).α-SMA-positive stromal myofibroblasts  cascade [β1+EGF  cells with TGF-gression , 61  [β1+EGF-stimulated HaCaT II-4 cells in a 3-dimensional system [TGF-lysin-2) , 73, whilysin-2)  is partilysin-2) –78. Whillysin-2) , where ilysin-2) . Despitel system . MMP-10,l system  creatingβ1+EGF [β1+EGF induces MMP-10 expression while dramatically enhancing PAI-1 production and stromal invasion [β1+EGF-stimulated HaCaT II-4 keratinocytes [β1+EGF activates MMPs-1, -7, -8, -9, and -13 stimulating plasmin-dependent matrix proteolysis. A corresponding upregulation of PAI-1 provides a sensitive focalized mechanism for titering the extent and duration of extracellular matrix degradation consequently sustaining a stromal scaffold necessary for tissue invasion. STAT3 in this context may promote this phenotype by regulating growth factor-dependent expression of critical remodeling factors such as MMP-10 and PAI-1  [srcc- is, in fact, a critical intermediate in a TGF-β1-initiated transduction cascade leading to MEK involvement, PAI-1 transcription, and downstream phenotypic responses [β1 complements EGF-mediated signaling to the MAPK/AKT pathways to effect EMT consistent with the requirement for oncogenic ras in TGF-β1-induced EMT [β1-stimulated ERK1/2 phosphorylation and PAI-1 transcription by src family kinase inhibitors, as well as blockade of EGFR signaling with AG1478, suggests that pp60srcc-, perhaps through phosphorylation of the Y845 src-kinase EGFR target residue, regulates MEK-ERK-dependent PAI-1 expression [β1-associated pp60srcc- kinase/EGFR stimulation remains to be determined, TGF-β1-dependent release of EGFR ligands  via MMP- or ADAM-dependent processes is one likely possibility for at least some cell types [β1-stimulated formation of integrin/FAK/p130cas/EGFR complexes may initiate ligand-independent EGFR activation and pp60srcc- recruitment [β1 transactivates the EGFR in a complex manner requiring src kinase signaling and production of reactive oxygen species but may not involve the shedding of EGFR ligands [β1-stimulated ERK1/2 phosphorylation and PAI-1 transcription by src kinase-targeting pharmacologic agents, as well as the EGFR inhibitor AG1478, and the requirement for MEK-ERK signaling for the full inductive effect of TGF-β1, suggests that pp60srcc-, perhaps through phosphorylation of the Y845 src-specific EGFR substrate residue regulates the MEK-ERK-dependent PAI-1 expression transduction cascade [β1-stimulated PAI-1 expression which provides novel insights as to the impact of TGF-β1 in late-stage human cutaneous tumor progression.In several common carcinoma types, including cutaneous SCC, the combination of TGF-henotype , 61, 80.se (MEK)  and the se (MEK) , 86–88. esponses , 86, 88.uced EMT , 90. Dispression , 85, 91 ll types . Alternaruitment , 94. Ind ligands . The eff cascade , 85, 86.β1 transcriptional responses due, at least in part, to the ability of MAP kinase-phosphorylated p53 to bind SMAD2 [Members of the p53 family are critical elements in a subset of TGF-nd SMAD2 –100. DNand SMAD2 . Two p53nd SMAD2 . p53-mednd SMAD2 .β1 since induced transcription is significantly attenuated in p53 siRNA knockdown cells [−/− mouse fibroblasts . This is consistent with the observation that p53-deficient lung tumor cells synthesize little or no PAI-1 while vector-engineered introduction of wild-type p53 rescues both basal and inducible PAI-1 expressions [β1 stimulation [ p53 is also required for maximal PAI-1 expression in response to TGF-wn cells  as well ressions . The recmulation , 104. Phmulation  which coβ1 [β1 expression during cutaneous SCC progression. The role of PAI-1 in subsequent tumor progression, however, may be more complex than previously appreciated. Ectopic expression of wild-type PAI-1 in breast cancer cells or in p53-deficient murine and human fibroblasts, in fact, initiates a senescence-like growth arrest [KD) or PAI-1 genetic deficiency (PAI-1−/− genotype) results in escape from replicative senescence in primary mouse and human fibroblasts [−/− endothelial cells, and PAI-1KD fibroblasts appears due, at least in part, to sustained activation of the PI3K-AKT-GSK3β pathway, increased AKTSer473 phosphorylation, nuclear retention of cyclin D1 [−/− mouse embryo fibroblasts (MEFs), PAI-1KD HaCaT keratinocytes, and PAI-1KD MEFs are resistant to TGF-β1-initiated growth inhibition although PAI-1 deficiency does not interfere with canonical TGF-β1 signaling such as SMAD phosphorylation or CIP1p21 and INK4Bp15 induction [p53 mutations occur in 40–60% of all skin cancers , 106 sugβ1  and may h arrest , 107 whiyclin D1  and, peryclin D1 . Importanduction . Collectively, these data suggest a multifunctional relationship between PAI-1 expression and tumor progression. Elevated PAI-1 levels may inhibit (at least transiently) tumor cell proliferation while stimulating migration and stromal invasion by providing a sensitive focalized mechanism for titering the extent and duration of extracellular matrix degradation, sustaining a stromal scaffold necessary for tissue invasion. This carefully orchestrated process may also serve to promote tumor cell survival by preventing anoikis during the precarious process of cell detachment and readhesion to a new, likely foreign, tissue microenvironment. Importantly, these findings underscore the potential diversity of new molecular targets that can be exploited for therapeutic benefit. Refining the current understanding of PAI-1 gene regulation, and relevant signaling pathways, may lead to the discovery of critical regulatory factors that ultimately prove important in stage-specific treatment of human cutaneous malignancies.


Caenorhabditis elegans' reproductive span is short relative to its total life span, with reproduction ceasing about a third into its 15–20 day adulthood. All of the known mutations and treatments that extend C. elegans' reproductive period also regulate longevity, suggesting that reproductive span is normally linked to life span. C. elegans has two canonical TGF-ß signaling pathways. We recently found that the TGF-ß Dauer pathway regulates longevity through the Insulin/IGF-1 Signaling (IIS) pathway; here we show that this pathway has a moderate effect on reproductive span. By contrast, TGF-ß Sma/Mab signaling mutants exhibit a substantially extended reproductive period, more than doubling reproductive span in some cases. Sma/Mab mutations extend reproductive span disproportionately to life span and act independently of known regulators of somatic aging, such as Insulin/IGF-1 Signaling and Dietary Restriction. This is the first discovery of a pathway that regulates reproductive span independently of longevity and the first identification of the TGF-ß Sma/Mab pathway as a regulator of reproductive aging. Our results suggest that longevity and reproductive span regulation can be uncoupled, although they appear to normally be linked through regulatory pathways.Female reproductive cessation is one of the earliest age-related declines humans experience, occurring in mid-adulthood. Similarly,  C. elegans and humans both have long post-reproductive life spans, leaving open the possibility that their reproductive spans might be extendable. C. elegans has been used previously to discover conserved regulators of aging, and here we use worms to identify a new regulator of reproductive aging, a highly conserved TGF-ß signaling pathway. We find that TGF-ß signaling regulates reproductive aging independently of somatic aging. This is the first identification of a pathway that breaks the coupling that normally links the two processes. Our work will provide new insights into the improvement of human fertility and prevention of age-related birth defects, and it has implications for the evolutionary relationship between reproduction and longevity regulation.Female reproductive cessation is the earliest aging phenotype humans experience, and its importance as a clinical issue is growing as more women opt to have children later in life. While much work has been done to understand the general aging process, little is currently known about the regulation of reproductive aging. Like longevity, the ability to produce progeny with advanced age is likely to be genetically regulated. Thus, understanding the processes that regulate reproductive aging may allow us to address the problems of maternal age-related infertility and birth defects.  Among human age-related declines, female reproductive cessation is one of the earliest to occur, with infertility and maternal age-related birth defects arising during the fourth decade of life C. elegans produces large broods of progeny and does not care for its young, its reproductive schedule is similar to human females' in that its fertility and reproduction sharply decline in early/mid-adulthood, followed by a long post-reproductive period C. elegans and human reproductive schedules suggest the intriguing possibility that studies in this model organism may reveal mechanisms regulating reproductive cessation across species.Although C. elegans studies have focused on reproductive fitness, measuring total numbers of offspring produced and average generation time, we are instead interested in identifying regulators of late-life reproduction . The standard assessments of fitness and fertility do not ascertain the length of time that an individual is capable of successful reproduction. However, we and others C. elegans ages significantly during the first week of adulthood, which is also reflected in the degree of germ line degeneration and oocyte quality decline While many C. elegans mutants currently known to delay reproductive aging were originally identified through their longevity phenotypes daf-2eat-2DrosophilaC. elegans hermaphrodites DrosophilaC. elegans shifts its reproductive strategy after starvation, modifying its production of males and its outcrossing frequency The C. elegans has two highly conserved Transforming Growth Factor-ß (TGF-ß) signaling pathways, the Dauer (daf-7) and Sma/Mab (dbl-1) pathways. We recently found that TGF-ß Dauer signaling regulates lifespan through its interactions with the Insulin/IGF-1 Signaling (IIS) pathway Small body/Male tail abnormal). The Sma/Mab pathway includes the ligand DBL-1, the type I receptor SMA-6, SMA-2 and SMA-3 R-Smads, the Co-Smad SMA-4, and the SMA-9 transcription co-factor  also increased reproductive span substantially  at the end of their reproductive period, in contrast to the Dauer mutants' very early egg-laying (Egl) and bagging phenotypes . In factC. elegans hermaphrodite sperm number limits wild-type self-fertilized reproduction, but mating with young (day 1 adult) males, whose sperm are not limited and outcompete those of the hermaphrodite, increases and prolongs progeny production fem-1) mutant hermaphrodites, which fail to make sperm when raised at restrictive temperatures. fem-1 mutants mated with wild-type males have a mean reproductive span of 6.3 days, while more than 90% of the mated sma-2;fem-1 double mutants were still fertile at day 12  and DR (eat-2) mutants In self-fertilized worms, sperm is only made prior to oogenesis C. elegans progeny number is linked to small body size via physical constraint of the maternal gonad and/or body size sma-9, is required in early larval development for the regulation of body size before gametogenesis sma-9 only in adulthood is sufficient to extend late reproduction , sma-1(ru18), sma-1(e30), dpy-1(e1) and dpy-9(e12)) whose body sizes are similar to the TGF-ß Sma/Mab mutants  produced only eggs that were able to hatch and develop to viable progeny , but on on day 7 , with ondaf-2 and eat-2 regulate reproductive aging C. elegans reproductive period also regulate longevity While dbl-1, sma-6) or inconsistently , while others appeared to have no effect on longevity   , S9, S10, sma-9) . While dife span . This efsma-2 mutants also increased with age at a similar rate as wild type , daf-16(mu86), and daf-16(mu86);daf-7(e1372) double mutants. We found that daf-7's reproductive span extension was significantly suppressed by loss of daf-16 activity  To tes mutants . Thus, weat-2-induced DR effect, which is caused by eat-2 mutants' inability to pump their pharynx to properly ingest food. However, we observed that over-expression of the DBL-1 ligand is not sufficient to suppress eat-2's reproductive span extension  in both worms and humans, these functions would all peak during the reproductive period, but begin to decline post-reproductively, each at a different rate. Successful reproduction requires peak physical condition of the soma; the increased matricide rates in older, reproductive Sma/Mab mutants shows that increased oocyte quality in the absence of healthy somatic tissues can be catastrophic, at least to the mother and any unproduced late progeny. By contrast, “survival” is the lowest measurable function (assayed as live vs. dead) and thus persists much longer than reproductive activity. Humans survive well past the age of peak physical function, a period that overlaps with female reproduction. Improvements in medicine, nutrition, hygiene, and environment have extended human life span significantly Longevity is regulated by insulin/IGF-1/FOXO signaling and by Dietary Restriction in worms through mammals C. elegans. If so, modulation of TGF-ß signaling may offer new avenues to improve fertility and offspring health in mothers of advanced age.Despite the vast differences in their life histories and chronological time frames, our work suggests that the regulation of worms and humans' longevity and reproductive spans may be conserved. Future studies will determine whether Sma/Mab mutants use a conserved mechanism to slow reproductive aging in daf-16(mu86), daf-8(e1393). LG II: eat-2(ad465), rrf-3(pk1426), sma-6(wk7). LG III: daf-2(e1370), daf-7(e1372), daf-4(e1364), sma-2(e502), sma-3(wk28), sma-3(wk20), sma-4(e729). LG IV: daf-1(m40), daf-14(m77), fem-1(hc17). LG V: dbl-1(nk3), dbl-1(wk70). LG X: sma-9(qc3), sma-9(wk55).All strains were cultured using standard methods ctIs40 X . CQ33: eat-2(ad465) II; ctIs40 X . CQ17: daf-1(m40) IV outcrossed to N2 3×. CQ16: daf-7(e1372) III outcrossed to N2 3×. CQ14: daf-14(m77) IV outcrossed to N2 3×. CQ19: sma-2(e502) III outcrossed to N2 3×. CQ18: sma-9(wk55) X outcrossed to N2 3×. CQ53: sma-2(e502);fem-1(hc17).Strains: BW1940: daf-16(mu86);sma-2(e502). CQ25: daf-16(mu86);daf-7(e1372). CF1041: daf-2(e1370) III.CQ49: sma-2(e502);fem-1(hc17) and fem-1(hc17) worms were shifted to 25°C from L3 and back to 20°C after L4. All experiments were performed with at least 10 individuals per strain . The log-rank (Mantel-Cox) method was used to test the null hypothesis. In mating reproductive span assays, L4 hermaphrodites were mated to young wild-type males at a 1∶3 ratio for 24 hours before being separated onto individual plates. Successful mating was ascertained by the fraction of male progeny each day. For the pha-4 RNAi reproductive span experiments, mothers were moved onto RNAi bacteria starting at L4.Individual synchronized L4 hermaphrodites were moved to fresh plates daily until reproduction ceased for at least two days. The last day of viable progeny production was noted as the day of reproduction cessation for each individual. When matricide occurred, the animal was censored from the experiment on that day. All experiments were performed at 20°C, except that Individual synchronized L4 hermaphrodites were moved to fresh plates and the number of progeny produced by each individual was counted daily until reproduction ceased for at least two days. When matricide occurred, the animal was censored from the experiment on that day. All experiments were performed at 20°C with at least 6 individuals per strain .The assay was performed as described in Reproductive Span analysis, except the cumulative percentage of hermaphrodites that underwent matricide was calculated daily. The matricide frequency was determined as the frequency of reproductive worms that die of matricide; as matricide is caused by internal progeny hatching, non-reproductive worms by definition never die of matricide, and thus are not included in calculation. This number reflects the likelihood of matricide.Eggs were synchronized by hypochlorite treatment and allowed to develop at 20°C until day 3 of adulthood. ∼100 synchronized hermaphrodites were transferred to a new plate and allowed to lay progeny for 1 hour; eggs and L1 progeny were counted at 3-hour intervals.The first day of adulthood was defined as t = 0, and the log-rank (Mantel-Cox) method was used to test the null hypothesis in Kaplan-Meier survival analysis, as previously described Bacterial feeding RNAi experiments were carried out as previously described pha-4 RNAi efficacy was determined by counting the arrested L1s produced by mothers fed from L4 onward compared with control vector.Figure S1C. elegans, with mouse homologs in parentheses.  Self-fertilized reproductive spans of TGF-β Dauer pathway mutants. (D) Percent increase in reproductive span of TGF-β Dauer pathway mutants over wild type. (E–G) Progeny production profiles of TGF-β Dauer pathway mutants. Statistics in TGF-β Dauer pathway mutants have only moderate effects on reproductive span. (A) Schematic representation of the TGF-β Dauer pathway in (1.56 MB TIF)Click here for additional data file.Figure S2C. elegans, with mouse homologs in parentheses.  Additional self-fertilized reproductive spans of the TGF-β Sma/Mab pathway mutants. (D) sma-2 and dbl-1 animals mated with wild-type males at day 4 still significantly extend reproductive span (p<0.0001 for each). (E) A higher percentage of Sma/Mab pathway mutants die of matricide than wild type.TGF-β Sma/Mab pathway regulates reproductive span. (A) Schematic representation of the TGF-β Sma/Mab pathway in (1.56 MB TIF)Click here for additional data file.Figure S3Progeny production profiles of TGF-β Sma/Mab pathway mutants. Self-fertilized animals (A–C) and wild-type mated animals .(1.56 MB TIF)Click here for additional data file.Figure S4sma-2 animals ovulate slower than wild type. (D–F) Mated sma-2, dbl-1, and sma-3 mutations reduce progeny production.TGF-β Sma/Mab mutants display small body size, slow ovulation, and reduced progeny number. (A) Example images comparing body size of wild type and three TGF-β Sma/Mab mutants. Scale bar represents 1 mm. (B) Average body length of genotypes in (A); mean±SEM. (C) Mated (1.56 MB TIF)Click here for additional data file.Figure S5sma-9 (Ahringer clone) RNAi whole-life and adult-only treated animals in late reproductive life. (B) Progeny number produced in late reproductive life, data from (A). (C) Last day of reproduction in sma-9 (Ahringer clone) RNAi whole-life and adult-only treated wild type or rrf-3 animals.TGF-β Sma/Mab pathway regulates late reproduction in adulthood. (A) Progeny production profile of (1.56 MB TIF)Click here for additional data file.Figure S6Five non-TGF-β small mutants. (A) Example images comparing body size of wild type and five other non-TGF-β small mutants. Scale bar represents 1 mm. (B) Average body length of genotypes in (A); mean±SEM.(1.56 MB TIF)Click here for additional data file.Figure S7daf-16(mu86) mutation suppresses sma-2's life span. (B) daf-16 and daf-16;sma-2 mutants increase matricide frequencies earlier. (C) daf-16 RNAi does not suppress sma-2's reproductive span. (D) daf-16 RNAi does not suppress dbl-1's reproductive span. Statistics in TGF-β Sma/Mab pathway mutants extend reproductive span independently of DAF-16/FOXO activity. (A) (1.56 MB TIF)Click here for additional data file.Figure S8sma-2;pha-4(RNAi) animals' body cavity are filled with unhatched eggs (white arrows). sma-2;pha-4(RNAi) animals have a severe egg-laying defect not observed in eat-2 or wild-type worms treated with pha-4 RNAi; many hermaphrodites were censored from the assay when their body cavity filled with arrested eggs, causing maternal death. This defect may have masked longer reproductive spans of the sma-2(e502);pha-4(RNAi) animals.(1.56 MB TIF)Click here for additional data file.Table S1TGF-β Sma/Mab pathway self-fertilized and wild-type mated reproductive spans.(0.10 MB PDF)Click here for additional data file.Table S2Self-fertilized reproductive spans (RS) of TGF-β Dauer pathway mutants.(0.09 MB PDF)Click here for additional data file.Table S3Self-fertilized brood size of TGF-β Sma/Mab pathway mutants.(0.09 MB PDF)Click here for additional data file.Table S4Mated brood size of TGF-β Sma/Mab pathway mutants.(0.09 MB PDF)Click here for additional data file.Table S5Mated reproductive spans (RS) of non-TGF-β small mutants.(0.07 MB PDF)Click here for additional data file.Table S6Life spans (LS) of TGF-β Sma/Mab pathway mutants.(0.09 MB PDF)Click here for additional data file.Table S7Comparison of life spans and self-fertilized reproductive spans in mutants with extended reproductive spans.(0.10 MB PDF)Click here for additional data file.Table S8Comparison of life spans and wild-type mated reproductive spans in mutants with extended reproductive spans.(0.08 MB PDF)Click here for additional data file.Table S9daf-16 loss-of-function on life spans and reproductive spans in TGF-β Dauer and Sma/Mab mutants.Effects of (0.10 MB PDF)Click here for additional data file.Table S10pha-4 loss-of-function on life spans and reproductive spans in eat-2 and TGF-β Sma/Mab mutants.Effects of (0.10 MB PDF)Click here for additional data file.Table S11dbl-1 over-expression on the reproductive span of eat-2 mutants.Effects of (0.07 MB PDF)Click here for additional data file.


Drosophila Pumilio and Caenorhabditis elegans FBF, both members of the PUF family translational regulators, contribute to GSC maintenance. FBF functions by suppressing GLD-1, which promotes meiotic entry. However, factors that directly promote GSC proliferation, rather than prevent differentiation, are not known. Here we show that PUF-8, another C. elegans member of the PUF family and MEX-3, a KH domain translational regulator, function redundantly to promote GSC mitosis. We find that PUF-8 protein is highly enriched in mitotic germ cells, which is similar to the expression pattern of MEX-3 described earlier. The puf-8(−) mex-3(−) double mutant gonads contain far fewer germ cells than both single mutants and wild-type. While these cells lack mitotic, meiotic and sperm markers, they retain the germ cell-specific P granules, and are capable of gametogenesis if GLP-1, which normally blocks meiotic entry, is removed. Significantly, we find that at least one of these two proteins is essential for germ cell proliferation even in meiotic entry-defective mutants, which otherwise produce germ cell tumors. We conclude PUF-8 and MEX-3 contribute to GSC maintenance by promoting mitotic proliferation rather than by blocking meiotic entry.Maintenance of mitotically cycling germline stem cells (GSCs) is vital for continuous production of gametes. In worms and insects, signaling from surrounding somatic cells play an essential role in the maintenance of GSCs by preventing premature differentiation. In addition, germ cell proteins such as the  Most sexually reproducing organisms generate gametes throughout the adult life. Presence of a population of mitotically cycling cells, called germline stem cells (GSCs), in their gonads enables the continuous generation of gametes. While self renewal ensures constant supply of germ cells, it alone is not sufficient to generate gametes: some of the mitotic cells must exit mitosis and differentiate into gametes. In addition, uncontrolled mitosis will lead to germ cell tumor. On the other hand, if all cells at some point enter meiosis, continuous generation of gametes will not be possible. What commits certain germ cells into differentiation and others into self renewal, and how this balance between mitosis and meiosis is maintained are some of the central questions in developmental biology.Caenorhabditis elegans GSC niche, formed by a single somatic cell called the distal tip cell (DTC), promotes germ cell proliferation by signaling through GLP-1, a LIN-12/Notch family receptor  mex-3(−) double mutant GSCs maintain germ cell character – they do differentiate into gametes if the meiotic block is removed – but neither proliferate nor enter into meiosis. Further, they are essential for the tumorous proliferation observed in meiotic defective mutants. Thus, PUF-8 and MEX-3 appear to directly promote GSC, rather than inhibit differentiation.Although different signaling pathways are engaged in the above three examples, suppression of differentiation clearly emerges as one common mechanism that controls GSC maintenance. However, blocking differentiation alone is unlikely to be sufficient to ensure self-renewal, for there are mutants, such as the ntiation . Therefounc-32(e189) glp-1(oz112)/unc-36(e251) glp-1(q175) III (BS913 — 175) III unc-13(e51) gld-1(q485)/hT2[dpy-18(h662)] I; +/hT2[bli-4(e937)] III (BS3156 — 37)] III fem-2(e2105)/unc-45(r450) dpy-1(e1) III (CB4035 — (e1) III glp-1(ar202) III (GC833 — 202) III +/nT1 IV; pos-1(zu148) unc-42(e270)/nT1 VJJ462 — mex-3(zu155) dpy-5(e61)/hT1 I; pos-1(zu148) unc-42(e270)/hT1 V (JJ1014 — 0)/hT1 V fog-2(q71) V (JK574 — 2(q71) V gld-2(q497) gld-1(q485)/hT2[qIs48]   gld-2(q497)/dpy-5(e61) unc-13(e51) I (JK1743 — 3(e51) I mex-3(zu155) dpy-5(e61)/hT1 I; puf-8(zh17) unc-4(e120)/mnC1 II; +/hT1 VIT21 — puf-8(zh17) unc-4(e120)/mnC1 IIIT60 — puf-8(zh17)unc-4(e120) II kpIs[pMP15]IT31 — mex-3(zu155) dpy-5(e61)/hT1 I; puf-8(zh17) unc-4(e120)/mnC1 II; glp-1(ar202) IIIIT95 — unc-13(e51) gld-1(q485)/hT2; puf-8(zh17) unc-4(e120)/mnC1 IIIT105 — mex-3(zu155) dpy-5(e61)/hT2[dpy-18(h662)] I; puf-8(zh17) unc-4(e120)/mnC1 II; glp-1(q175)/hT2[bli-4(e937)] IIIIT83 — mex-3(zu155) dpy-5(e61) gld-2(q497) gld-1(q485)/hT1 I; puf-8(zh17) unc-4(e120)/mnC1 IIIT80 — mex-3(zu155) dpy-5(e61)/hT1 I; puf-8(zh17) unc-4(e120)/mnC1 II; fem-2(e2105)/unc-45(r450) dpy-1(e1) III; +/hT1 VIT111 — puf-8(zh17) unc-4(e120)/mnC1 II; fog-2(q71) VIT57 — fog-2(q71) V; mex-3(zu155) dpy-5(e61)/hT1 I; puf-8(zh17) unc-4(e120)/mnC1 II; +/hT1 VIT116 — mex-3(zu155) dpy-5(e61)/hT2[dpy-18(h662)] I; puf-8(zh17) unc-4(e120)/mnC1 II; glp-1(oz112)/hT2[bli-4(e937)] IIIIT113 — bnIs1[pie-1::GFP::pgl-1 + unc-119(+)] (SS747 — -119(+)] Worms strains were maintained as described , except pos-1, IT21 worms were crossed with JJ462 males and F1 progeny cloned to see no progeny gave 100% dead embryo (n = 30).Generation of IT21: JJ1104 were crossed with IT60 males and resulting F1 progeny were crossed among themselves. The F2 progeny worms were placed one worm per plate (hereafter referred to as “cloning”) and the worm with the relevant phenotype was selected to get IT21. In order to rule out the presence of delinked mex-3(zu155) dpy-5(e61)/hT1 I; puf-8(zh17) unc-4(e120)/mnC1 II, which were obtained during this cross, were allowed to loose mex-3 and dumpy (Dpy) worms were screened for the presence of GFP::PGL-1. Once Dpy worms with GFP were obtained, they were made homozygous for it by crossing with the genotype GFP::PGL-1 dpy-5(e61)/hT1 I; puf-8(zh17) unc-4(e120)/mnC1 II. These worms were crossed with IT21 males and the resultant F1 Dpy worms were again crossed with IT21 males. F2 progeny were then cloned to get IT21 genotype along with GFP. Presence of mex-3(zu155) was ascertained by selecting Dpy worms that produced 100% dead embryos.To incorporate GFP::PGL-1 into IT21 background, IT21 males were crossed with SS747 and the F1 males were crossed back with IT21. F2 hermaphrodites were cloned to get IT21 genotype as well as GFP; however, no such worm was found indicating that GFP::PGL-1 insertion may be on chromosome I. GFP::PGL-1/mex-3(zu155), IT83 worms were crossed with IT21 males and F1 non-sterile Dpy progeny were cloned and selected based on the production of 100% dead embryos.BS913 worms were crossed with IT21 males; F1 males were crossed with hT2 Dpy hermaphrodites coming from IT105. F2 progeny were selected for presence of the desired genotype. These worms would produce sterile Dpy progeny; to confirm the presence of GC833 worms were crossed with IT21 males and F1 males were crossed back to IT21 hermaphrodites. F2 worms were cloned and embryos were collected in two sets, one at 15 °C and the other 25 °C for each cloned worm. The plate with IT21 genotype was identified based on the presence of the tumor phenotype among non-Unc worms at 25 °C. Embryos were again collected in 2 sets from the sibling plate kept at 15 °C. IT95 phenotype was identified based on 100% tumor phenotype at 25 °C; the siblings incubated at 15 °C were selected for maintaining IT95.dpy-5(e61) unc-13(e51) I) from JK1743 were crossed with IT60 males and these were crossed with IT89 worms and F2 cloned. Plates having the genotype dpy-5(e61) unc-13(e51)/ gld-2(q497) gld-1(q485) I; puf-8(zh17) unc-4(e120)/mnC1 II were selected. The recombinant Dpy non-Unc worms were cloned. These worms were crossed with IT21 to incorporate hT1 balancer and were labeled as IT93. Presence of gld-2(q497) and gld-1(q485) were confirmed by complementation with JK2879 strain. The IT93 worms were crossed with IT21 males and F1 non-sterile Dpy worms were crossed back with IT21 hermaphrodites. F2 worms were cloned and worms showing the genotype of IT80 were selected. mex-3(zu155), gld-2(q497) and gld-1(q485) were all again confirmed by complementation.First IT89 was made by crossing JK2879 hermaphrodites with IT21 males and F2 males were crossed back with IT21 hermaphrodites. F2 were cloned and the plate with the desired genotype was selected. Dpy Unc worms (fem-2(e2105).Hermaphrodites and Dpy Unc worms from CB4035 were mated separately with IT21 males, and the F1 males from one cross plate were mated with hermaphrodites from the other. F2 worms were cloned to obtain IT111 genotype. From such selected plates F3 worms were again cloned and observed for maternal-effect feminization, which confirmed the presence of JK574 females were crossed with IT60 and F1 males were crossed back with IT60 hermaphrodites. F2 were cloned and plates with feminized Unc worms were selected. Males from these plates were generated and also hermaphrodites were cloned to maintain the line. Multiple single-male crosses were set with feminized hermaphrodites from the maintained line. Plates in which 100% of the hermaphrodites were feminized were selected as IT57.Procedure to generate this strain was similar to that of IT57, except that the first cross here used IT57 females and IT21 males.puf-8(zh17) unc-4(e120)/mnC1 males; these males were then crossed with BS913 worms. The resulting progeny were clones to get worms with the genotype, +/hT2I; puf-8(zh17) unc-4(e120)/+ II; glp-1(oz112)/hT2 III. These worms were crossed with IT83 males and the resulting progeny were cloned to get the desired genotype of IT113.hT2 worms from IT105 were crossed with IT60 males to get +/hT2; puf-8 was PCR-amplified as a single piece from C. elegans genomic DNA and cloned between Kpn I and Eco RI sites of Bluescript KS+ vector (Stratagene). In the resulting construct, two kb of sequences immediately downstream of puf-8 stop codon was PCR-amplified from the genomic DNA and cloned between Not I and Sac II sites. The coding sequence of GFP was then PCR-amplified from pKS111HisΔ5 (unc-119 rescuing sequences were PCR-amplified from pKS111HisΔ5 and inserted at the Sac II site.The plasmid construct pMP15 carrying the PUF-8::GFP transgene was generated in the following manner. Two kb upstream sequences and the coding region of 111HisΔ5  and clonunc-119(−) strain by biolistic bombardment as described (puf-8 (−) mutant phenotype of IT60 upon mating. Both the rescued strains, IT31 and IT32, showed identical GFP expression pattern.The resulting plasmid, pMP15, was introduced into escribed . Of the Dissection and fixation of gonads for staining with the DNA-binding dye, 4,6-diamidino-2-phenylindole (DAPI), were performed as described earlier . For immStained gonads were mounted in Vectashield (Vector laboratories), examined using a fluorescence microscope (Zeiss Axioskop II mot plus) and imaged using a CCD camera (Axiocam HRm). GFP fluorescence was also examined and imaged similarly.puf-8(−) gonad is shorter and contains fewer germ cells than wild-type (puf-8(zh17), which is a strong loss-of-function allele (puf-8. Two independent transgenic lines carrying this transgene were obtained and both successfully rescued the phenotype of puf-8(zh17). The expression patterns of GFP in both these lines were identical. GFP was first observed in the descendants of the primordial germ cells in the early L1 larva. While GFP fluorescence was observed in the cytoplasm, stronger, punctuate fluorescence around the nucleus was unmistakable. This expression pattern continued through L2 and L3 stages. At L4 and adult stages, GFP expression was strongest in the mitotic germ cells and gradually decreased as the germ cells progressed through meiosis  mex-3(−) double mutant worms and compared the number of germ cells in them with the wild-type and both single mutants. At 20 °C, all mex-3(−) worms (n = 132) were fertile and produced dead embryos, consistent with its known function in embryogenesis (puf-8(−) worms, 99% (n = 139) of them were fertile. However, all the double mutant worms were sterile (n = 300) and did not produce any embryos. We visualized the germ cell nuclei in the dissected gonads of 1-day old adults by staining with the DNA-binding dye DAPI and counted these nuclei. The number of germ cells in both single mutants were somewhat reduced: in mex-3(−) gonads it was about 61%, and in puf-8(−) gonads it was about 35% of the wild-type. In contrast, the double mutant contained far fewer germ cells (10% of wild-type)   and no gpuf-8(−) mex-3(−) double mutant. Observation of GFP fluorescence revealed that the punctate, perinuclear arrangement of P granules, which is characteristic of wild-type germ cells, was unaffected in the mutant larvae. However, the number of germ cells in the double mutant L2 larva was significantly less than the wild-type. In later larval stages, while the germ cells increased dramatically in the wild-type, they showed only a marginal increase in the double mutant. Late stage wild-type L4 larva contained about 240 germ cells/gonad, whereas this number for the same age double mutant larva was only about 24 , and REC-8, a protein involved in sister chromatid cohesion (puf-8(−) mex-3(−) animals and immunostained them with anti-PH3 and anti-REC-8 antibodies. As shown in puf-8(−) and mex-3(−) single mutant gonads. In contrast, the chromatin of the double mutant germ cells was not REC-8-positive. Instead, only a weak REC-8 staining was observed in the nucleoplasm. Consistently, PH3-positive cells were observed only in the wild-type and single mutant gonads  mex-3(−) animals was due to lack of GLP-1, we immunostained their gonads with anti-GLP-1 antibody. Surprisingly, the level of GLP-1 protein in the double mutant gonad was similar to the wild-type. However, its distribution pattern in the double mutant was markedly different from the wild-type and both the single mutants (glp-1(−) puf-8(−) mex-3(−) triple mutants and examined their germ cells by DAPI staining. As shown in glp-1(−) single mutant. These results show that the double mutant germ cells are capable of meiosis and gametogenesis, and their inability to enter meiosis is due to the presence of functional GLP-1 in them. Their meiotic potential, when taken together with the presence of intact P granules in them, strongly suggests that these cells were indeed germ cells, and have not entered into somatic differentiation.Expression of GLP-1, a receptor protein of the LIN-12/Notch family, by GSCs is essential for their mitotic proliferation. In the absence of GLP-1, all germ cells enter into meiosis and form sperm . The prie absent . To test mutants B. In theBased on the results presented in this section, we conclude PUF-8 and MEX-3 are not essential for germ cell identity or entry into meiosis.glp-1(−) indicates that the puf-8(−) mex-3(−) double mutant germ cells have not differentiated, to further validate this observation, we checked for the expression of meiotic as well as sperm markers in these cells. The synaptonemal complex protein HIM-3 and the sperm-specific membranous organelle (MO) serve as good markers for meiosis and sperm, respectively (puf-8(−) and mex-3(−) single mutants, the double mutant gonads did not contain HIM-3- or MO-positive cells  and gld-1(−) gld-2(−) mutants should be dependent on the presence of PUF-8 or MEX-3. We tested this possibility by examining germ cell proliferation in puf-8(−) mex-3(−) glp-1(gf) triple and puf-8(−) mex-3(−) gld-1(−) gld-2(−) quadruple mutants. As we had predicted, these triple and the quadruple mutant combinations did not produce germ cell tumors. Instead, they had roughly the same number of germ cells as the puf-8(−) mex-3(−) double mutant (puf-8(−) mex-3(−) double mutant, and not either single mutant, was epistatic over the glp-1(gf) single mutant and the gld-1(−) gld-2(−) double mutants  mex-3(−) germ cells did not enter differentiation and did not proliferate even when the meiotic entry was blocked. We conclude PUF-8 and MEX-3 do not suppress differentiation in GSCs, but promote mitosis, regardless of the status of differentiation signals.In summary, the puf-8(+/−) mex-3(−/−) and puf-8(−/−) mex-3(+/−) mutants, which carry a single dose of only one of the two proteins, and examined the status of germ cell development in them. Worms carrying only one copy of puf-8 were essentially identical to the wild-type. Mitotic proliferation was normal in these worms and they produced both types of gametes. Worms carrying a single copy of mex-3 had a smaller germline, consistent with the phenotype of the puf-8(−/−) single mutant. Surprisingly, germ cells in about 34% of these worms differentiated only as sperm, indicating that they were defective for the sperm/oocyte switch  mex-3(+/−) produce only sperm, we made triple mutant combinations with two mutants, namely fem-2(−) and fog-2(−), which produce oocytes only. Both puf-8(−) mex-3(+/−) fem-2(−) and puf-8(−) mex-3(+/−) fog-2(−) triple mutants produced only oocytes (puf-8(−) mex-3(+/−) is dependent on the activities of both fem-2 and fog-2. Since fog-2 is known to act upstream of fem-2, our results place puf-8 and mex-3 ahead of fog-2 in the hermaphrodite germline sex determination pathway.ogenesis . We perf oocytes , indicatpuf-8(−) mex-3(−) double mutant gonads contain greatly reduced number of germ cells. Similar to normal GSCs, the double mutant germ cells possess perinuclear P granules and lack both meiotic and sperm markers. Importantly, they are able to differentiate into sperm, if GLP-1, which mediates meiotic suppression in GSCs, is removed. These observations clearly show that these cells are indeed germ cells and that PUF-8 and MEX-3 are not required to maintain germ cell identity or to suppress meiosis. However, the double mutant germ cells show several mitotic defects. Their chromosomes lack REC-8, which is normally present in proliferative germ cells. In addition, as revealed by immunostaining with the phospho-histone H3 antibody, the double mutant gonads do not contain any cell in mitotic metaphase. These observations strongly suggest a role for PUF-8 and MEX-3 in the mitotic competence of GSCs. Thus, these two proteins are not required to suppress differentiation, but are essential for proliferation.Earlier studies on germline stem cell (GSC) maintenance have unearthed mechanisms that suppress premature differentiation. In this study, we provide evidence for the existence of mechanism(s) that promote GSC mitosis. Our results reveal that PUF-8 and MEX-3 function redundantly to promote mitosis in germline stem cells (GSCs). Compared to the wild-type and the single mutants, the puf-8 and mex-3 are expressed in the mitotic region and encode RNA-binding proteins [puf-8(−) mex-3(−) double mutant germ cells show several mitotic defects, germline-specific cell cycle regulators constitute a potential group of common targets for PUF-8 and MEX-3. Germline-specific members are known for at least a few cell cycle regulators. One among them, cdc25.1, encodes the C. elegans homolog of CDC25 phosphatase, which activates cyclin-dependent kinase (CDK)  and the CDK inhibitory protein CKI-1 (Both se (CDK) . Disruptlt gonad . Other pin CKI-1 .Drosophila PUF protein Pumilio, which depends on Nanos for the translational control of hunchback mRNA  fbf-1(−/−) and puf-8(−/−) mex-3(−/+) double mutants is the reduction in the size of the mitotic region. Further, in a synthetic screen for mutants that display germline phenotype in puf-8(−/−) background, we have recently isolated several mutations that are defective for both sperm/oocyte switch and normal level of proliferation . Taken together, these observations indicate a link between the size of the mitotic region and the sperm/oocyte switch, and support the earlier proposal (tra-2 and fem-3.We find that PUF-8 and MEX-3 function redundantly in sperm/oocyte switch, and their requirement is genetically upstream of the known genes of the sex determination pathway. Intriguingly, PUF-8 functions redundantly with FBF-1 as well in sperm/oocyte switch, which again has been shown to be upstream of the other known genes of the sex determination pathway . How do ogenesis . Howeverogenesis , and FBFnslation , both ofproposal  that the