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Short- and long-term CDDP responses at low (2 mg/kg) and high (5 mg/kg) doses of CDDP were characterized in xenografted tumors

Impact of some RNAi clones on cisplatin toxicity in Caenorhabditis elegans adults

Purpose: To investigate the genetic basis of cisplatin resistance as efficacy of cisplatin-based chemotherapy in the treatment of distinct malignancies is often hampered by intrinsic or acquired drug resistance of tumor cells.Experimental Design: We produced 14 orthoxenograft transplanting human nonseminomatous testicular germ cell tumors (TGCT) in mice, keeping the primary tumor features in terms of genotype, phenotype, and sensitivity to cisplatin. Chromosomal and genetic alterations were evaluated in matched cisplatin-sensitive and their counterpart orthoxenografts that developed resistance to cisplatin in nude mice.Results: Comparative genomic hybridization analyses of four matched orthoxenografts identified recurrent chromosomal rearrangements across cisplatin-resistant tumors in three of them, showing gains at 9q32-q33.1 region. We found a clinical correlation between the presence of 9q32-q33.1 gains in cisplatin-refractory patients and poorer overall survival (OS) in metastatic germ cell tumors. We studied the expression profile of the 60 genes located at that genomic region. POLE3 and AKNA were the only two genes deregulated in resistant tumors harboring the 9q32-q33.1 gain. Moreover, other four genes (GCS, ZNF883, CTR1, and FLJ31713) were deregulated in all five resistant tumors independently of the 9q32-q33.1 amplification. RT-PCRs in tumors and functional analyses in Caenorhabditis elegans (C. elegans) indicate that the influence of 9q32-q33.1 genes in cisplatin resistance can be driven by either up- or downregulation. We focused on glucosylceramide synthase (GCS) to demonstrate that the GCS inhibitor DL-threo-PDMP resensitizes cisplatin-resistant germline-derived orthoxenografts to cisplatin.Conclusions: Orthoxenografts can be used preclinically not only to test the efficiency of drugs but also to identify prognosis markers and gene alterations acting as drivers of the acquired cisplatin resistance. Clin Cancer Res; 24(15); 3755–66. ©2018 AACR.

Supplementary materials and methods, supplementary tables and supplementary figure legends

Representative recurrent imbalanced regions in different orthoxenografts with acquired CDDP resistance

Physical and Functional interactions of proteins encoded by 9q32-q33.1 genes expressed in PDOXs of this study

Comparative histological analysis of original non-treated orthoxenografts and their pairs with acquired resistance to CDDP after five cycles of treatment in mice

Regulated protein clearance is vital for cells to maintain protein homeostasis and the conditions essential for survival. The primary machinery for intracellular protein degradation is the ubiquitin– proteasome system (UPS), by which ubiquitin-tagged proteins are degraded by the proteasome. Proteasomes are present both in the cytoplasm and the nucleus, but the mechanisms coordinating proteasome activity and its subcellular localization in a multicellular organism are still unclear. Here, we identified the nuclear protein-encoding geneakir-1as a proteasome regulator in a genome-wideCaenorhabditis elegans(C. elegans) RNAi screen. We show that the depletion ofakir-1causes accumulation of endogenous polyubiquitinated proteins in the nuclei of intestinal cells, concomitant with slowerin vivoproteasomal degradation in this subcellular compartment. Remarkably, the loss ofakir-1does not induce an accumulation of polyubiquitinated proteins in oocyte nuclei, thoughakir-1is essential for the nuclear localization of proteasomes in both cell types. We further show that the importin family memberima-3genetically interacts withakir-1, and affects subcellular distribution of polyubiquitinated proteins in intestinal cells. We show for the first time that conserved AKIR-1 is important for the nuclear transport of proteasomes in a multicellular organism, suggesting a role for AKIR-1 in the maintenance of proteostasis.

The Cas9 nuclease from Streptococcus pyogenes (SpCas9) is the most popular enzyme for CRISPR technologies. However, considering the wide diversity of microorganisms (discovered and still unknown), a massive number of CRISPR effectors are being and will be identified and characterized in the search of optimal Cas variants for each of the many applications of CRISPR. In this context, a versatile and efficient multicellular system for CRISPR editing such as Caenorhabditis elegans would be of great help in the development of these effectors. Here, we highlight the benefits of using C. elegans for the rapid evaluation of new CRISPR effectors, and for optimizing CRISPR efficiency in animals in several ways such as by modulating the balance between repair pathways, modifying chromatin accessibility, or controlling the expression and activity of nucleases and guide RNAs.

Context Genes causing familial forms of diabetes mellitus are only partially known. Objective We set out to identify the genetic cause of hyperglycemia in multigenerational families with an apparent autosomal dominant form of adult-onset diabetes not due to mutations in known monogenic diabetes genes. Methods Existing whole-exome sequencing (WES) data were used to identify exonic variants segregating with diabetes in 60 families from the United States and Italy. Functional studies were carried out in vitro (transduced MIN6-K8 cells) and in vivo (Caenorhabditis elegans) to assess the diabetogenic potential of 2 variants in the malate dehydrogenase 2 (MDH2) gene linked with hyperglycemia in 2 of the families. Results A very rare mutation (p.Arg52Cys) in MDH2 strongly segregated with hyperglycemia in 1 family from the United States. An infrequent MDH2 missense variant (p.Val160Met) also showed disease cosegregation in a family from Italy, although with reduced penetrance. In silico, both Arg52Cys and Val160Met were shown to affect MDH2 protein structure and function. In transfected HepG2 cells, both variants significantly increased MDH2 enzymatic activity, thereby decreasing the NAD+/NADH ratio—a change known to affect insulin signaling and secretion. Stable expression of human wild-type MDH2 in MIN6-K8 cell lines enhanced glucose- and GLP-1-stimulated insulin secretion. This effect was blunted by the Cys52 or Met160 substitutions. Nematodes carrying equivalent changes at the orthologous positions of the mdh-2 gene showed impaired glucose-stimulated insulin secretion. Conclusion Our findings suggest a central role of MDH2 in human glucose homeostasis and indicate that gain of function variants in this gene may be involved in the etiology of familial forms of diabetes.

The requirement for Cas nucleases to recognize a specific PAM is a major restriction for genome editing. SpCas9 variants SpG and SpRY, recognizing NGN and NRN PAM, respectively, have contributed to increase the number of editable genomic sites in cell cultures and plants. However, their use has not been demonstrated in animals.We have characterized and optimized the activity of SpG and SpRY in zebrafish and C. elegans. Delivered as mRNA-gRNA or ribonucleoprotein (RNP) complexes, SpG and SpRY were able to induce mutations in vivo, albeit at a lower rate than SpCas9 in equivalent formulations. This lower activity was overcome by optimizing mRNA-gRNA or RNP concentration, leading to efficient mutagenesis at regions inaccessible to SpCas9. We also found that the CRISPRscan algorithm can predict SpG and SpRY activity in vivo. Finally, we applied SpG and SpRY to generate knock-ins by homology-directed repair. Altogether, our results expand the CRISPR-Cas targeting genomic landscape in animals.

We previously showed that fluorizoline, a fluorinated thiazoline compound, binds to both subunits of the mitochondrial prohibitin (PHB) complex, PHB1 and PHB2, being the expression of these proteins required for fluorizoline-induced apoptosis in mouse embryonic fibroblasts. To investigate the conservation of this apoptotic mechanism, we studied the effect of PHB downregulation on fluorizoline activity on two human cell lines, HEK293T and U2OS. Then, we asked whether PHBs mediate the effect of fluorizoline in a multicellular organism. Interestingly, reduced levels of PHBs in the human cells impaired the induction of apoptosis by fluorizoline. We observed that fluorizoline has a detrimental dose-dependent effect on the development and survival of the nematode model Caenorhabditis elegans. Besides, such effects of fluorizoline treatment in living nematodes were absent in PHB mutants. Finally, we further explored the apoptotic pathway triggered by fluorizoline in human cell lines. We found that the BH3-only proteins NOXA, BIM and PUMA participate in fluorizoline-induced apoptosis and that the induction of NOXA and PUMA is dependent on PHB expression., This work was supported by grants from the Agencia Estatal de Investigación (Ministerio de Ciencia e Innovación), European Regional Development Fund (ERDF), the European Research Council, the Junta de Andalucía and the Instituto de Salud Carlos III (ISCIII) (SAF2017-83178-R to J.G.; PID2019-107991RB-I00 to R.L.; ERC-2011-StG-281691 and C2A ID: 42571/Exp: 70806 to M.A-S; PI15-00895 to J.C.). J.S-E and I.S-V are recipients of research fellowships from the Ministerio de Ciencia e Innovación. S.N-V is recipient of a research fellowship from Universitat de Barcelona. MD.M-B was supported by the Plan de Empleo Juvenil (EJP09) from the Junta de Anadalucía. D.K has a FI AGAUR fellowship from Generalitat de Catalunya.

We previously showed that fluorizoline, a fluorinated thiazoline compound, binds to both subunits of the mitochondrial prohibitin (PHB) complex, PHB1 and PHB2, being the expression of these proteins required for fluorizoline-induced apoptosis in mouse embryonic fibroblasts. To investigate the conservation of this apoptotic mechanism, we studied the effect of PHB downregulation on fluorizoline activity on two human cell lines, HEK293T and U2OS. Then, we asked whether PHBs mediate the effect of fluorizoline in a multicellular organism. Interestingly, reduced levels of PHBs in the human cells impaired the induction of apoptosis by fluorizoline. We observed that fluorizoline has a detrimental dose-dependent effect on the development and survival of the nematode model Caenorhabditis elegans. Besides, such effects of fluorizoline treatment in living nematodes were absent in PHB mutants. Finally, we further explored the apoptotic pathway triggered by fluorizoline in human cell lines. We found that the BH3-only proteins NOXA, BIM and PUMA participate in fluorizoline-induced apoptosis and that the induction of NOXA and PUMA is dependent on PHB expression.

SummaryMammalian IκB proteins (IκBs) exert their main function as negative regulators of NF-κB, a central signaling pathway controlling immunity and inflammation. An alternative chromatin role for IκBs has been shown to affect stemness and cell differentiation. However, the involvement of NF-κB in this function has not been excluded. NFKI-1 and IKB-1 are IκB homologs inCaenorhabditis elegans, which lacks NF-κB nuclear effectors. We found thatnfki-1andikb-1mutants display developmental defects that phenocopy mutations in Polycomb and UTX-1 histone demethylase, suggesting a role forC. elegansIκBs in chromatin regulation. Further supporting this possibility(i)we detected NFKI-1 in the nucleus of cells;(ii)NFKI-1 and IKB-1 bind to histones and Polycomb proteins,(iii)and associate with chromatinin vivo, and(iv)mutations innfki-1andikb-1alter chromatin marks. Based on these results, we propose that ancestral IκB inhibitors modulate Polycomb activity at specific gene subsets with an impact on development.

The transcriptomes of model organisms have been defined under specific laboratory growth conditions. The standard protocol for Caenorhabditis elegans growth and maintenance is 20°C on an Escherichia coli diet. Temperatures ranging from 15°C to 25°C or feeding with other species of bacteria are considered physiological conditions, but the effect of these conditions on the worm transcriptome has not been well characterized. Here, we compare the global gene expression profile for the reference Caenorhabditis elegans strain (N2) grown at 15°C, 20°C, and 25°C on two different diets, Escherichia coli and Bacillus subtilis. When C. elegans were fed E. coli and the growth temperature was increased, we observed an enhancement of defense response pathways and down-regulation of genes associated with metabolic functions. However, when C. elegans were fed B. subtilis and the growth temperature was increased, the nematodes exhibited a decrease in defense response pathways and an enhancement of expression of genes associated with metabolic functions. Our results show that C. elegans undergo significant metabolic and defense response changes when the maintenance temperature fluctuates within the physiological range and that the degree of pathogenicity of the bacterial diet can further alter the worm transcriptome. © Gómez-Orte et al.

Notch signaling is an evolutionarily conserved signal transduction pathway that is essential for metazoan development. Upon ligand binding, the Notch intracellular domain (NOTCH ICD) translocates into the nucleus and forms a complex with the transcription factor RBPJ (also known as CBF1 or CSL) to activate expression of Notch target genes. In the absence of a Notch signal, RBPJ acts as a transcriptional repressor. Using a proteomic approach, we identified L3MBTL3 (also known as MBT1) as a novel RBPJ interactor. L3MBTL3 competes with NOTCH ICD for binding to RBPJ. In the absence of NOTCH ICD, RBPJ recruits L3MBTL3 and the histone demethylase KDM1A (also known as LSD1) to the enhancers of Notch target genes, leading to H3K4me2 demethylation and to transcriptional repression. Importantly, in vivo analyses of the homologs of RBPJ and L3MBTL3 in Drosophila melanogaster and Caenorhabditis elegans demonstrate that the functional link between RBPJ and L3MBTL3 is evolutionarily conserved, thus identifying L3MBTL3 as a universal modulator of Notch signaling in metazoans.

Neurexins and neuroligins are neuronal membrane adhesion molecules that have been involved in neuropsychiatric and neurodevelopmental disorders. The nrx-1 and nlg-1 genes of Caenorhabditis elegans encode NRX-1 and NLG-1, orthologue proteins of human neurexins and neuroligins, respectively. Dopaminergic and serotoninergic signalling control the locomotory rate of the nematode. When well-fed animals are transferred to a plate with food (bacterial lawn), they reduce the locomotory rate. This behavior, which depends on dopamine, is known as basal slowing response (BSR). Alternatively, when food-deprived animals are moved to a plate with a bacterial lawn, further decrease their locomotory rate. This behavior, known as enhanced slowing response (ESR), is serotonin dependent. C. elegans nlg-1-deficient mutants are impaired in BSR and ESR. Here we report that nrx-1-deficient mutants were defective in ESR, but not in BSR. The nrx-1;nlg-1 double mutant was impaired in both behaviors. Interestingly, the nlg-1 mutants upregulate the expression of comt-4 which encodes an enzyme with putative catechol-O-methyltransferase activity involved in dopamine degradation. Our study also shows that comt-4(RNAi) in nlg-1-deficient mutants rescues the wild type phenotypes of BSR and ESR. On the other hand, comt-4(RNAi) in nlg-1-deficient mutants also recovers, at least partially, the gentle touch response and the pharyngeal pumping rate that were impaired in these mutants. These latter behaviors are dopamine and serotonin dependent, respectively. Based on these results we propose a model for the neuroligin function in modulating the dopamine-dependent locomotory behavior in the nematode.

CRISPR/Cas and the high conservation of the spliceosome components facilitate the mimicking of human pathological mutations in splicing factors of model organisms. The degenerative retinal disease retinitis pigmentosa (RP) is caused by mutations in distinct types of genes, including missense mutations in splicing factors that provoke RP in an autosomal dominant form (s-adRP). Using CRISPR in Caenorhabditis elegans, we generated mutant strains to mimic s-adRP mutations reported in PRPF8 and SNRNP200. Whereas these inherited mutations are present in heterozygosis in patients, C. elegans allows the maintenance of these mutations as homozygotes, which is advantageous for genetic and drug screens. We found that snrp-200(cer23[V676L]) and prp-8(cer14[H2302del]) display pleiotropic phenotypes, including reduced fertility. However, snrp-200(cer24[S1080L]) and prp-8(cer22[R2303G]) are weak alleles suitable for RNAi screens for identifying genetic interactions, which could uncover potential disease modifiers. We screened a collection of RNAi clones for splicing-related genes and identified three splicing factors: isy-1/ISY1, cyn-15/PPWD1 and mog-2/SNRPA1, whose partial inactivation may modify the course of the disease. Interestingly, these three genes act as modifiers of prp-8(cer22) but not of snrp-200(cer24). Finally, a screen of the strong allele prp-8(cer14) with FDA-approved drugs did not identify molecules capable of alleviating the temperature-sensitive sterility. Instead, we detected drugs, such as dequalinium chloride, which exacerbated the phenotype, and therefore, are potentially harmful to s-adRP patients since they may accelerate the progression of the disease.

CRISPR and the high conservation of the spliceosome components facilitate the mimicking of human pathological mutations in splicing factors of model organisms. The degenerative retinal disease Retinitis Pigmentosa (RP) is caused by mutations in distinct types of genes, including missense mutations in splicing factors that provoke RP in an autosomal dominant form (s-adRP). Using CRISPR in C. elegans, we generated mutant strains to mimic RP mutations reported in PRPF8 and SNRNP200. Whereas these inherited mutations are present in heterozygosis in patients, C. elegans allows the maintenance of these mutations in homozygosis, which is advantageous for genetic and drug screens. We found that snrp-200(cer23[V676L]) and prp-8(cer14[H2302del]) display pleiotropic phenotypes, including a reduced fertility. However, snrp-200(cer24[S1080L]) and prp-8(cer22[R2303G]) are weak alleles suitable for RNAi screens to identify genetic interactions, which would uncover potential disease modifiers. We screened a collection of RNAi clones for splicing-related genes and identified three splicing factors, isy-1/ISY1, cyn-15/PPWD1 and mog-2/SNRPA1 whose partial inactivation may modify the course of the disease. Interestingly, these three genes were acting as modifiers of prp-8(cer22) but no snrp-200(cer24).Finally, the strong allele prp-8(cer14) was used in a screen with FDA-approved drugs to find molecules capable of alleviating the phenotype. Instead, we detected drugs, as Dequalinium Chloride, which exacerbated the phenotype and therefore are potentially harmful for s-adRP patients since they may accelerate the progression of the disease.

The authors uncover a role for the proteostasis modulator AIRAPL as a tumor suppressor in myeloproliferative malignancies, through its regulation of IGFR stability. The results ascribe a biological function to AIRAPL, and they implicate prosteostatic deregulation as an oncogenic mechanism in myeloid transformation, thus suggesting potential novel therapeutic strategies. AIRAPL (arsenite-inducible RNA-associated protein-like) is an evolutionarily conserved regulator of cellular proteostasis linked to longevity in nematodes, but its biological function in mammals is unknown1,2,3. We show herein that AIRAPL-deficient mice develop a fully-penetrant myeloproliferative neoplastic process. Proteomic analysis of AIRAPL-deficient mice revealed that this protein exerts its antineoplastic function through the regulation of the insulin/insulin-like growth factor 1 (IGF-1) signaling pathway. We demonstrate that AIRAPL interacts with newly synthesized insulin-related growth factor-1 receptor (IGF1R) polypeptides, promoting their ubiquitination and proteasome-mediated degradation. Accordingly, genetic and pharmacological IGF1R inhibitory strategies prevent the hematological disease found in AIRAPL-deficient mice as well as that in mice carrying the Jak2V617F mutation, thereby demonstrating the causal involvement of this pathway in the pathogenesis of myeloproliferative neoplasms4,5,6. Consistent with its proposed role as a tumor suppressor of myeloid transformation, AIRAPL expression is widely abrogated in human myeloproliferative disorders. Collectively, these findings support the oncogenic relevance of proteostasis deregulation in hematopoietic cells, and they unveil novel therapeutic targets for these frequent hematological neoplasias.

CRISPR-based genome-editing methods in model organisms are evolving at an extraordinary speed. Whereas the generation of deletion or missense mutants is quite straightforward, the production of endogenous fluorescent reporters is more challenging. We have developed Nested CRISPR, a cloning-free ribonucleoprotein-driven method that robustly produces endogenous fluorescent reporters with EGFP, mCherry or wrmScarlet in Caenorhabditis elegans. This method is based on the division of the fluorescent protein (FP) sequence in three fragments. In the first step, single-stranded DNA (ssDNA) donors (≤200 bp) are used to insert the 5′ and 3′ fragments of the FP in the locus of interest. In the second step, these sequences act as homology regions for homology-directed repair using a double-stranded DNA (dsDNA) donor (PCR product) containing the middle fragment, thus completing the FP sequence. In Nested CRISPR, the first step involving ssDNA donors is a well-established method that yields high editing efficiencies, and the second step is reliable because it uses universal CRISPR RNAs (crRNAs) and PCR products. We have also used Nested CRISPR in a nonessential gene to produce a deletion mutant in the first step and a transcriptional reporter in the second step. In the search for modifications to optimize the method, we tested synthetic single guide RNAs (sgRNAs), but did not observe a significant increase in efficiency. To streamline the approach, we combined all step 1 and step 2 reagents in a single injection and were successful in three of five loci tested with editing efficiencies of up to 20%. Finally, we discuss the prospects of this method in the future.

CRISPR-based genome editing methods in model organisms are evolving at an extraordinary speed. Whereas the generation of deletion or missense mutants is quite straightforward, the production of endogenous fluorescent reporters is still inefficient. The use of plasmids with selection markers is an effective methodology, but often requires laborious and complicated cloning steps. We have established a cloning-free ribonucleoprotein-driven Nested CRISPR method that robustly produces endogenous fluorescent reporters. This methodology is based on the division of the GFP and mCherry sequences in three fragments. In the first step we use ssDNA donors (≤200 bp) to insert 5’ and 3’ fragments in the place of interest. In the second step, we use these sequences as homology regions for Homology Directed Repair (HDR) with a dsDNA donor (PCR product, ≈700 bp) including the middle fragment, thus completing the fluorescent protein sequence. This method is advantageous because the first step with ssDNA donors is known to be very efficient, and the second step, uses universal reagents, including validated PCR products and crRNAs, to create fluorescent reporters reaching reliable editing efficiencies as high as 40%. We have also used Nested CRISPR in a non-essential gene to produce a deletion mutant in the first step and a transcriptional reporter in the second step.In the search of modifications to optimize the method, we tested synthetic sgRNAs, but we did not observe a significant increase in the efficacy compared to independently adding tracrRNA and crRNA to the injection mix. Conveniently, we also found that both steps of Nested CRISPR could be performed in a single injection. Finally, we discuss the utility of Nested CRISPR for targeted insertion of long DNA fragments in other systems and prospects of this method in the future.

[Purpose] To investigate the genetic basis of cisplatin resistance as efficacy of cisplatin-based chemotherapy in the treatment of distinct malignancies is often hampered by intrinsic or acquired drug resistance of tumor cells., [Experimental Design] We produced 14 orthoxenograft transplanting human nonseminomatous testicular germ cell tumors (TGCT) in mice, keeping the primary tumor features in terms of genotype, phenotype, and sensitivity to cisplatin. Chromosomal and genetic alterations were evaluated in matched cisplatin-sensitive and their counterpart orthoxenografts that developed resistance to cisplatin in nude mice., [Results] Comparative genomic hybridization analyses of four matched orthoxenografts identified recurrent chromosomal rearrangements across cisplatin-resistant tumors in three of them, showing gains at 9q32-q33.1 region. We found a clinical correlation between the presence of 9q32-q33.1 gains in cisplatin-refractory patients and poorer overall survival (OS) in metastatic germ cell tumors. We studied the expression profile of the 60 genes located at that genomic region. POLE3 and AKNA were the only two genes deregulated in resistant tumors harboring the 9q32-q33.1 gain. Moreover, other four genes (GCS, ZNF883, CTR1, and FLJ31713) were deregulated in all five resistant tumors independently of the 9q32-q33.1 amplification. RT-PCRs in tumors and functional analyses in Caenorhabditis elegans (C. elegans) indicate that the influence of 9q32-q33.1 genes in cisplatin resistance can be driven by either up- or downregulation. We focused on glucosylceramide synthase (GCS) to demonstrate that the GCS inhibitor DL-threo-PDMP resensitizes cisplatin-resistant germline-derived orthoxenografts to cisplatin, [Conclusions] Orthoxenografts can be used preclinically not only to test the efficiency of drugs but also to identify prognosis markers and gene alterations acting as drivers of the acquired cisplatin resistance., Several authors are grateful recipients of predoctoral fellowships: J.M. Piulats from the AECC and F.J. García-Rodríguez from the Instituto de Salud Carlos III (ISCIII). This study was supported by grants from the Spanish Ministry of Economy and Competitiveness (SAF2002-02265 and FIS: BFU2007-67123; PI10-0222, PI13-01339, and PI16/01898, to A. Villanueva; PI15-00895, to J. Ceron; SAF2013-46063R, to F. Vi nals; PI030264, to ~ X. García-del-Muro), Fundacio La Marat o TV3 (051430, to F. Vi nals and X. ~ García-del-Muro), Generalitat de Catalunya (2014SGR364, to A. Villanueva and F. Vinals; FIS09/0059, to A. Morales), cofunded by FEDER funds/ ~ European Regional Development Fund (ERDF) — a way to Build Europe. A. Villanueva received a BAE11/00073 grant. We thank the staff of the Animal Core Facility of IDIBELL for mouse care and maintenance.

Cisplatin and derivatives are commonly used as chemotherapeutic agents. Although the cytotoxic action of cisplatin on cancer cells is very efficient, clinical oncologists need to deal with two major difficulties, namely the onset of resistance to the drug and the cytotoxic effect in patients. Here, we used Caenorhabditis elegans to investigate factors influencing the response to cisplatin in multicellular organisms. In this hermaphroditic model organism, we observed that sperm failure is a major cause of cisplatin-induced infertility. RNA sequencing data indicate that cisplatin triggers a systemic stress response, in which DAF-16/FOXO and SKN-1/NRF2, two conserved transcription factors, are key regulators. We determined that inhibition of the DNA damage-induced apoptotic pathway does not confer cisplatin protection to the animal. However, mutants for the pro-apoptotic BH3-only gene ced-13 are sensitive to cisplatin, suggesting a protective role of the intrinsic apoptotic pathway. Finally, we demonstrated that our system can also be used to identify mutations providing resistance to cisplatin and therefore potential biomarkers of innate cisplatin-refractory patients. We show that mutants for the redox regulator trxr-1, ortholog of the mammalian thioredoxin reductase 1 TRXR1, display cisplatin resistance. By CRISPR/Cas9, we determined that such resistance relies on the presence of the single selenocysteine residue in TRXR-1., This study was supported by grants from the Instituto de Salud Carlos III (ISCIII; PI15/00895 to J.C., PI16/01898 to A.V.), which is co-funded by the European Regional Development Fund/FEDER. The A.V. laboratory was also supported by funding from Ministerio de Economía y Competitividad (BFU2007-67123). M.B. was supported by a Netherlands Organization for Scientific Research (NWO) chemical sciences ECHO grant (711.014.005). E.N. was supported a Sociedad Española de Oncología Médica (SEOM) grant for emerging research groups. F.J.G.-R. was the recipient of a PFIS predoctoral fellowship from ISCIII. A.M.-V. was supported by a grant from Ministerio de Economía y Competitividad (BFU2015-64408-P, co-financed by the European Social Fund) and is a member of the EU-ROS Cost Action of the European Union. J.C., M.B., S.H. and A.M.-V are members of the GENIE EU Cost action. D.B. was recipient of a Consejo Nacional de Ciencia y Tecnología ‘al extranjero’ fellowship (523048).

Cisplatin and derivatives are commonly used as chemotherapeutic agents. Although the cytotoxic action of cisplatin on cancer cells is very efficient, clinical oncologists need to deal with two major difficulties: (i) the onset of resistance to the drug, and (ii) the cytotoxic effect in patients. Here we useCaenorhabditis elegansto investigate factors influencing the response to cisplatin in multicellular organisms. In this hermaphroditic model organism, we observed that sperm failure is a major cause in cisplatin-induced infertility. RNA-seq data indicate that cisplatin triggers a systemic stress response in which DAF-16/FOXO and SKN-1/Nrf2, two conserved transcription factors, are key regulators. We determined that inhibition of the DNA-damage induced apoptotic pathway does not confer cisplatin protection to the animal. However, mutants for the pro-apoptotic BH3-only geneced-13are sensitive to cisplatin, suggesting a protective role of the intrinsic apoptotic pathway. Finally, we demonstrate that our system can also be used to identify mutations providing resistance to cisplatin and therefore potential biomarkers of innate cisplatin-refractory patients. We show that mutants for the redox regulatortrxr-1, ortholog of the mammalian Thioredoxin-Reductase-1 TrxR1, display cisplatin resistance and that such resistance relies on a single selenocysteine residue.

The transcriptomes of model organisms have been defined under specific laboratory growth conditions. The standard protocol for Caenorhabditis elegans growth and maintenance is 20°C on an Escherichia coli diet. Temperatures ranging from 15°C to 25°C or feeding with other species of bacteria are considered physiological conditions, but the effect of these conditions on the worm transcriptome has not been well characterized. Here, we compare the global gene expression profile for the reference Caenorhabditis elegans strain (N2) grown at 15°C, 20°C, and 25°C on two different diets, Escherichia coli and Bacillus subtilis. When C. elegans were fed E. coli and the growth temperature was increased, we observed an enhancement of defense response pathways and down-regulation of genes associated with metabolic functions. However, when C. elegans were fed B. subtilis and the growth temperature was increased, the nematodes exhibited a decrease in defense response pathways and an enhancement of expression of genes associated with metabolic functions. Our results show that C. elegans undergo significant metabolic and defense response changes when the maintenance temperature fluctuates within the physiological range and that the degree of pathogenicity of the bacterial diet can further alter the worm transcriptome.

The Scientific Foundation ‘Asociación Española Contra el Cáncer’ (AECC, Stable Coordinated Group, Hereditary Cancer); the BBVA Foundation; the Eugenio Rodríguez Pascual Foundation grant 2012; Generalitat de Catalunya AGAUR SGR 2012 grants 283, 290 and 312, and SGR 2014 grants 364, 530, and 535; Spanish Ministry of Health ISCIII FIS grants PI10/00057, PI10/00222, PI10/01422, PI12/01528, PI13/00132, and PI14/00336. ISCIII RTICC grants RD06/0020/1051, RD12/0036/0007, RD12/0036/0008 and RD12/0036/0063; Spanish Ministry of Science and Innovation, ‘Fondo Europeo de Desarrollo Regional (FEDER), una manera de hacer Europa’, MINECO grants SAF2010-20203 and SAF2013-46196; and the Telemaraton 2014 ‘Todos Somos Raros, Todos Somos Únicos’ grant P35 (...), Mateo, F.; Arenas, E. J.; Aguilar, H.; Serra-Musach, J.; de Garibay, G. Ruiz; Boni, J.; Maicas, M.; Du, S.; Iorio, F.; Herranz-Ors, C.; Islam, A.; Prado, X.; Llorente, A.; Petit, A.; Vidal, A.; Catala, I.; Soler, T.; Venturas, G.; Rojo-Sebastian, A.; Serra, H.; Cuadras, D.; Blanco, I.; Lozano, J.; Canals, F.; Sieuwerts, A. M.; de Weerd, V.; Look, M. P.; Puertas, S.; Garcia, N.; Perkins, A. S.; Bonifaci, N.; Skowron, M.; Gomez-Baldo, L.; Hernandez, V.; Martinez-Aranda, A.; Martinez-Iniesta, M.; Serrat, X.; Ceron, J.; Brunet, J.; Barretina, M. P.; Gil, M.; Falo, C.; Fernandez, A.; Morilla, I.; Pernas, S.; Pla, M. J.; Andreu, X.; Segui, M. A.; Ballester, R.; Castella, E.; Nellist, M.; Morales, S.; Valls, J.; Velasco, A.; Matias-Guiu, X.; Figueras, A.; Sanchez-Mut, J. V.; Sanchez-Cespedes, M.; Cordero, A.; Gomez-Miragaya, J.; Palomero, L.; Gomez, A.; Gajewski, T. F.; Cohen, E. E. W.; Jesiotr, M.; Bodnar, L.; Quintela-Fandino, M.; Lopez-Bigas, N.; Valdes-Mas, R.; Puente, X. S.; Vinals, F.; Casanovas, O.; Graupera, M.; Hernandez-Losa, J.; Ramon y Cajal, S.; Garcia-Alonso, L.; Saez-Rodriguez, J.; Esteller, M.; Sierra, A.; Martin-Martin, N.; Matheu, A.; Carracedo, A.; Gonzalez-Suarez, E.; Nanjundan, M.; Cortes, J.; Lazaro, C.; Odero, M. D.; Martens, J. W. M.; Moreno-Bueno, G.; Barcellos-Hoff, M. H.; Villanueva, A.; Gomis, R. R.; Pujana, M. A.

Includes supplementary materials for the online appendix. Many eukaryotic genes contain embedded antisense transcripts and repetitive sequences of unknown function. We report that male germline-specific expression of an antisense transcript contained in an intron of C. elegans Target of Rapamycin (TOR, let-363) is associated with (1) accumulation of endo-small interfering RNAs (siRNAs) against an embedded Helitron transposon and (2) activation of an alternative 3′ splice site of TOR. The germline-specific Argonaute proteins PRG-1 and CSR-1, which participate in self/nonself RNA recognition, antagonistically regulate the generation of these endo-siRNAs, TOR mRNA levels, and 3′ splice-site selection. Supply of exogenous double-stranded RNA against the region of sense/antisense overlap reverses changes in TOR expression and splicing and suppresses the progressive multigenerational sterility phenotype of prg-1 mutants. We propose that recognition of a “nonself” intronic transposon by endo-siRNAs/the piRNA system provides physiological regulation of expression and alternative splicing of a host gene that, in turn, contributes to the maintenance of germline function across generations. We thank A. Fire, S. Ahmed, and E. Miska for sharing information and helpful discussions, members of our laboratories for critical feedback, the Caenorhabditis Genetics Center (CGC) for providing strains, Matthew Ragle for collection of sperm samples, Alan Zahler for assistance and critical feedback, and Montserrat Porta de la Riva for technical assistance. S.B.-S. was supported by an EMBO postdoctoral fellowship. A.T.L. is a Taub Fellow, supported by the Taub Foundation. J.C. is a Miguel Servet Junior Investigator. Work in J.V.’s lab is supported by Fundación Botín, Consolider RNAREG and Ministerio de Economía y Competitividad (MINECO). Work in B.L.’s lab is supported by the European Research Council (ERC), MINECO Plan Nacional grants BFU2008-00365 and BFU2011-26206, ERASysBio+ ERANET project EUI2009-04059 GRAPPLE, the EMBO Young Investigator Program, EU Framework 7 project 277899 4DCellFate, the AXA Research Fund, and the EMBL/CRG Systems Biology Program. Support from the CRG Genomics Facility is also acknowledged.

SWI/SNF ATP-dependent chromatin-remodeling complexes have been related to several cellular processes such as transcription, regulation of chromosomal stability, and DNA repair. The Caenorhabditis elegans gene ham-3 (also known as swsn-2.1) and its paralog swsn-2.2 encode accessory subunits of SWI/SNF complexes. Using RNA interference (RNAi) assays and diverse alleles we investigated whether ham-3 and swsn-2.2 have different functions during C. elegans development since they encode proteins that are probably mutually exclusive in a given SWI/SNF complex. We found that ham-3 and swsn-2.2 display similar functions in vulva specification, germline development, and intestinal cell proliferation, but have distinct roles in embryonic development. Accordingly, we detected functional redundancy in some developmental processes and demonstrated by RNA sequencing of RNAi-treated L4 animals that ham-3 and swsn-2.2 regulate the expression of a common subset of genes but also have specific targets. Cell lineage analyses in the embryo revealed hyper-proliferation of intestinal cells in ham-3 null mutants whereas swsn-2.2 is required for proper cell divisions. Using a proteomic approach, we identified SWSN-2.2-interacting proteins needed for early cell divisions, such as SAO-1 and ATX-2, and also nuclear envelope proteins such as MEL-28. swsn-2.2 mutants phenocopy mel-28 loss-of-function, and we observed that SWSN-2.2 and MEL-28 colocalize in mitotic and meiotic chromosomes. Moreover, we demonstrated that SWSN-2.2 is required for correct chromosome segregation and nuclear reassembly after mitosis including recruitment of MEL-28 to the nuclear periphery., Some strains were provided by the Caenorhabditis Genetics Center, which is funded by National Institutes of Health Office of Research Infrastructure Programs (P40 OD010440). I.E. was supported by an IDIBELL (Bellvitge Biomedical Research Institute) Ph.D. fellowship. J.C. is a Miguel Servet Researcher (ISCIII). This study was supported by a grant from the Instituto de Salud Carlos III (ISCIII) (PI12/01554), which is cofunded by FEDER funds/European Regional Development Fund—a way to build Europe. The P.A. laboratory was supported by funding from the Spanish Ministry of Economy and Competitiveness (BFU2013-42709-P).

A key aim of neurodevelopmental research is to understand how precursor cells decide to stop dividing and commence their terminal differentiation at the correct time and place. Here, we show that minibrain (mnb), the Drosophila ortholog of the Down syndrome candidate gene DYRK1A, is transiently expressed in newborn neuronal precursors known as ganglion cells (GCs). Mnb promotes the cell cycle exit of GCs through a dual mechanism that regulates the expression of the cyclin-dependent kinase inhibitor Dacapo, the homolog of vertebrate p27(Kip1) (Cdkn1b). Mnb upregulates the expression of the proneural transcription factor (TF) Asense, which promotes Dacapo expression. Mnb also induces the expression of Prospero, a homeodomain TF that in turn inhibits the expression of Deadpan, a pan-neural TF that represses dacapo In addition to its effects on Asense and Prospero, Mnb also promotes the expression of the neuronal-specific RNA regulator Elav, strongly suggesting that Mnb facilitates neuronal differentiation. These actions of Mnb ensure the precise timing of neuronal birth, coupling the mechanisms that regulate neurogenesis, cell cycle control and terminal differentiation of neurons., This work was supported by grants from the Dirección General de Investigación Científica y Técnica [BFU2009-08831, BFU2012-38892 to F.J.T.]; the Generalitat Valenciana [ACOMP/2012/047 to F.J.T.]; and the Fundación Inocente Inocente [050666100002 to F.J.T.]. J. Ceron and J. Colonques were recipients of PhD fellowships from the Ministerio de Educación y Ciencia. M.N.S. was recipient of a Santiago Grisolia Fellowship from the Generalitat Valenciana.

Colorectal cancer is the second cause of cancer-related death in the western world, and although the genetic and molecular mechanisms involved in the initiation and progression of these tumors are among the best characterized, there are significant gaps in our understanding of this disease. The role of EPHB signaling in colorectal cancer has only recently been realized. Here, we use animal models to investigate the role of EphB4 in intestinal tumorigenesis. Modulation of EPHB4 levels in colon cancer cell lines resulted in significant differences in tumor growth in a xenograft model, with low levels of EPHB4 associated with faster growth. In addition, using a genetic model of intestinal tumorigenesis where adenomatous polyposis coli (Apc) mutations lead to initiation of the tumorigenic process (Apcmin mice), we show that inactivation of a single allele of EphB4 results in higher proliferation in both the normal epithelium and intestinal tumors, significantly larger tumors in the small intestine, and a 10-fold increase in the number of tumors in the large intestine. This was associated with a 25% reduction in the lifespan of Apcmin mice (P < 0.0001). Gene expression analysis showed that EphB4 mutations result in a profound transcriptional reprogramming, affecting genes involved in cell proliferation, remodeling of the extracellular matrix, and cell attachment to the basement membrane among other functional groups of genes. Importantly, in agreement with the expression profiling experiments, using an in vitro assay, we show that loss of EPHB4 in colon cancer cells results in a significantly increased potential to invade through a complex extracellular matrix. Collectively, these results indicate that EphB4 has tumor suppressor activities and that regulation of cell proliferation, extracellular matrix remodeling, and invasive potential are important mechanisms of tumor suppression. [Cancer Res 2009;69(18):7430–8]

Mdx mice show a milder phenotype than Duchenne patients despite bearing an analogous genetic defect. Our aim was to sort out genes, differentially expressed during the evolution of skeletal muscle mdx mouse disease, to elucidate the mechanisms by which these animals overcome the lack of dystrophin. Genome-wide microarray-based gene expression analysis was carried out at 3 wk and 1.5 and 3 mo of life. Candidate genes were selected by comparing: 1) mdx vs. controls at each point in time, and 2) mdx mice and 3) control mice among the three points in time. The first analysis showed a strong upregulation (96%) of inflammation-related genes and in >75% of genes related to cell adhesion, muscle structure/regeneration, and extracellular matrix remodeling during mdx disease evolution. Lgals3, Postn, Ctss, and Sln genes showed the strongest variations. The analysis performed among points in time demonstrated significant changes in Ecm1, Spon1, Thbs1, Csrp3, Myo10, Pde4b, and Adamts-5 exclusively during mdx mice lifespan. RT-PCR analysis of Postn, Sln, Ctss, Thbs1, Ecm1, and Adamts-5 expression from 3 wk to 9 mo, confirmed microarray data and demonstrated variations beyond 3 mo of age. A high-confidence functional network analysis demonstrated a strong relationship between them and showed two main subnetworks, having Dmd- Utrn- Myo10 and Adamts5- Thbs1- Spon1-Postn as principal nodes, which are functionally linked to Abca1, Actn4, Crebbp, Csrp3, Lama1, Lama3, Mical2, Mical3, Myf6, Pxn, and Sparc genes. Candidate genes may participate in the decline of muscle necrosis in mdx mice and could be considered potential therapeutic targets for Duchenne patients.

Due to its intermediate complexity and its sophisticated genetic tools, the larval brain of Drosophila is a useful experimental system to study the mechanisms that control the generation of cell diversity in the CNS. In order to gain insight into the neuronal and glial lineage specificity of neural progenitor cells during postembryonic brain development, we have carried an extensive mosaic analysis throughout larval brain development. In contrast to embryonic CNS development, we have found that most postembryonic neurons and glial cells of the optic lobe and central brain originate from segregated progenitors. Our analysis also provides relevant information about the origin and proliferation patterns of several postembryonic lineages such as the superficial glia and the medial-anterior Medulla neuropile glia. Additionally, we have studied the spatio-temporal relationship between gcm expression and gliogenesis. We found that gcm expression is restricted to the post-mitotic cells of a few neuronal and glial lineages and it is mostly absent from postembryonic progenitors. Thus, in contrast to its major gliogenic role in the embryo, the function of gcm during postembryonic brain development seems to have evolved to the specification and differentiation of certain neuronal and glial lineages.

Although glial cells missing (gcm) genes are known as glial determinants in the fly embryo, the role of vertebrate orthologs in the central nervous system is still under debate. Here we show for the first time that the chicken ortholog of fly gcm (herein referred to as c-Gcm1), is expressed in early neuronal lineages of the developing spinal cord and is required for neural progenitors to differentiate as neurons. Moreover, c-Gcm1 overexpression is sufficient to trigger cell cycle exit and neuronal differentiation in neural progenitors. Thus, c-Gcm1 expression constitutes a crucial step in the developmental cascade that prompts progenitors to generate neurons: c-Gcm1 acts downstream of proneural (neurogenin) and progenitor (Sox1-3) factors and upstream of NeuroM neuronal differentiation factor. Strikingly, this neurogenic role is not specific to the vertebrate gene, as fly gcmand gcm2 are also sufficient to induce the expression of neuronal markers. Interestingly, the neurogenic role is restricted to post-embryonic stages and we identify two novel brain neuronal lineages expressing and requiring gcm genes. Finally, we show that fly gcm and the chick and mouse orthologs induce expression of neural markers in HeLa cells. These data, which demonstrate a conserved neurogenic role for Gcm transcription factors, call for a re-evaluation of the mode of action of these genes during evolution.

Retinitis pigmentosa (RP) is a rare genetic disease that causes gradual blindness through retinal degeneration. Intriguingly, seven of the 24 genes identified as responsible for the autosomal-dominant form (adRP) are ubiquitous spliceosome components whose impairment causes disease only in the retina. The fact that these proteins are essential in all organisms hampers genetic, genomic, and physiological studies, but we addressed these difficulties by using RNAi in Caenorhabditis elegans. Our study of worm phenotypes produced by RNAi of splicing-related adRP (s-adRP) genes functionally distinguishes between components of U4 and U5 snRNP complexes, because knockdown of U5 proteins produces a stronger phenotype. RNA-seq analyses of worms where s-adRP genes were partially inactivated by RNAi, revealed mild intron retention in developing animals but not in adults, suggesting a positive correlation between intron retention and transcriptional activity. Interestingly, RNAi of s-adRP genes produces an increase in the expression of atl-1 (homolog of human ATR), which is normally activated in response to replicative stress and certain DNA-damaging agents. The up-regulation of atl-1 correlates with the ectopic expression of the pro-apoptotic gene egl-1 and apoptosis in hypodermal cells, which produce the cuticle, but not in other cell types. Our model in C. elegans resembles s-adRP in two aspects: The phenotype caused by global knockdown of s-adRP genes is cell type-specific and associated with high transcriptional activity. Finally, along with a reduced production of mature transcripts, we propose a model in which the retina-specific cell death in s-adRP patients can be induced through genomic instability.

The recently completed Caenorhabditis elegans genome sequence allows application of high-throughput (HT) approaches for phenotypic analyses using RNA interference (RNAi). As large phenotypic data sets become available, “phenoclustering” strategies can be used to begin understanding the complex molecular networks involved in development and other biological processes. The current HT-RNAi resources represent a great asset for phenotypic profiling but are limited by lack of flexibility. For instance, existing resources do not take advantage of the latest improvements in RNAi technology, such as inducible hairpin RNAi. Here we show that a C. elegans ORFeome resource, generated with the Gateway cloning system, can be used as a starting point to generate alternative HT-RNAi resources with enhanced flexibility. The versatility inherent to the Gateway system suggests that additional HT-RNAi libraries can now be readily generated to perform gene knockdowns under various conditions, increasing the possibilities for phenome mapping in C. elegans.

The Minibrain (Mnb) gene belongs to a new protein kinase family, which is evolutionarily conserved, and probably plays several roles during brain development and in adulthood. In Drosophila, mnb is involved in postembryonic neurogenesis and in learning/memory. In humans, MNB has been mapped within the Down syndrome critical region of chromosome 21 and is overexpressed in the Down syndrome embryonic brain. It has been widely proposed that MNB is involved in the neurobiological alterations associated with Down syndrome. Nevertheless, little is known about the functional role that MNB plays in vertebrate brain development. We have recently shown [Hammerle et al. (2002) Dev. Biol., 246, 259-273] that in early vertebrate embryos, Mnb is transiently expressed in neural progenitor cells during the transition from proliferating to neurogenic divisions. Here we have studied in detail a second wave of Mnb expression, which takes place in the brain of intermediate and late vertebrate embryos. In these stages, MNB seems to be restricted to certain populations of neurons, as no consistent expression was detected in astroglial or oligodendroglial cells. Interestingly, MNB expression takes place at the time of dendritic tree differentiation and is initiated by a transient translocation from the cytoplasm to the nucleus. Afterwards, MNB protein is transported to the growing dendritic tree, where it colocalizes with Dynamin 1, a putative substrate of MNB kinases. We propose that MNB kinase is involved in the signalling mechanisms that regulate dendrite differentiation. This functional role helps to build a new hypothesis for the implication of MNB/DYRK1A in the developmental aetiology of Down syndrome neuropathologies.

A mathematical model that describes the oscillatory dynamic experimentally observed in the volumetric flows of CO2/O2 during photosynthesis is used in order to study the response of the photosynthetic process to changes in the external temperature. The model allows modeling steady, oscillatory and damped transitions between states, in relation the flows of matter and the substrate concentrations, but in order to study the effect of temperature, we added the energy balance equation to the model and we took the entire photosynthetic process to the scale of a reactor chloroplast. Variation in external temperature is carried out in different ways and. in order to analyze the photosynthetic model’s response to thermal changes; we choose the variation in the generation of entropy as the second law criteria. Results show that entropy generated during the heating process is specific to the way it’s carried out and that the system reacts more efficiently in response to a Fourier heating.

This is an open-access article distributed under the terms of the Creative Commons Attribution License.-- et al., Protein components of the spliceosome are highly conserved in eukaryotes and can influence several steps of the gene expression process. RSR-2, the Caenorhabditis elegans ortholog of the human spliceosomal protein SRm300/SRRM2, is essential for viability, in contrast to the yeast ortholog Cwc21p. We took advantage of mutants and RNA interference (RNAi) to study rsr-2 functions in C. elegans, and through genetic epistasis analysis found that rsr-2 is within the germline sex determination pathway. Intriguingly, transcriptome analyses of rsr-2(RNAi) animals did not reveal appreciable splicing defects but instead a slight global decrease in transcript levels. We further investigated this effect in transcription and observed that RSR-2 colocalizes with DNA in germline nuclei and coprecipitates with chromatin, displaying a ChIP-Seq profile similar to that obtained for the RNA Polymerase II (RNAPII). Consistent with a novel transcription function we demonstrate that the recruitment of RSR-2 to chromatin is splicing-independent and that RSR-2 interacts with RNAPII and affects RNAPII phosphorylation states. Proteomic analyses identified proteins associated with RSR-2 that are involved in different gene expression steps, including RNA metabolism and transcription with PRP-8 and PRP-19 being the strongest interacting partners. PRP-8 is a core component of the spliceosome and PRP-19 is the core component of the PRP19 complex, which interacts with RNAPII and is necessary for full transcriptional activity. Taken together, our study proposes that RSR-2 is a multifunctional protein whose role in transcription influences C. elegans development. © 2013 Fontrodona et al., This work was funded by the European Commision (Marie Curie International Reintegration Grant: MIRG-CT-2007-206584), the Carlos III Institute (FIS: PS09/02145, PI12/01554), and the Marató of TV3 (Ref. 100910). Laura Fontrodona was supported by an AGAUR predoctoral fellowship. Julián Cerón is a Miguel Servet Junior Investigator.

Research into the molecular and developmental biology of the nematode Caenorhabditis elegans was begun in the early seventies by Sydney Brenner and it has since been used extensively as a model organism. C. elegans possesses key attributes such as simplicity, transparency and short life cycle that have made it a suitable experimental system for fundamental biological studies for many years. Discoveries in this nematode have broad implications because many cellular and molecular processes that control animal development are evolutionary conserved. C. elegans life cycle goes through an embryonic stage and four larval stages before animals reach adulthood. Development can take 2 to 4 days depending on the temperature. In each of the stages several characteristic traits can be observed. The knowledge of its complete cell lineage together with the deep annotation of its genome turn this nematode into a great model in fields as diverse as the neurobiology, aging, stem cell biology and germ line biology. An additional feature that makes C. elegans an attractive model to work with is the possibility of obtaining populations of worms synchronized at a specific stage through a relatively easy protocol. The ease of maintaining and propagating this nematode added to the possibility of synchronization provide a powerful tool to obtain large amounts of worms, which can be used for a wide variety of small or high-throughput experiments such as RNAi screens, microarrays, massive sequencing, immunoblot or in situ hybridization, among others. Because of its transparency, C. elegans structures can be distinguished under the microscope using Differential Interference Contrast microscopy, also known as Nomarski microscopy. The use of a fluorescent DNA binder, DAPI (4',6-diamidino-2-phenylindole), for instance, can lead to the specific identification and localization of individual cells, as well as subcellular structures/defects associated to them.

Research into the molecular and developmental biology of the nematode Caenorhabditis elegans was begun in the early seventies by Sydney Brenner and it has since been used extensively as a model organism. C. elegans possesses key attributes such as simplicity, transparency and short life cycle that have made it a suitable experimental system for fundamental biological studies for many years. Discoveries in this nematode have broad implications because many cellular and molecular processes that control animal development are evolutionary conserved. C. elegans life cycle goes through an embryonic stage and four larval stages before animals reach adulthood. Development can take 2 to 4 days depending on the temperature. In each of the stages several characteristic traits can be observed. The knowledge of its complete cell lineage together with the deep annotation of its genome turn this nematode into a great model in fields as diverse as the neurobiology, aging, stem cell biology and germ line biology. An additional feature that makes C. elegans an attractive model to work with is the possibility of obtaining populations of worms synchronized at a specific stage through a relatively easy protocol. The ease of maintaining and propagating this nematode added to the possibility of synchronization provide a powerful tool to obtain large amounts of worms, which can be used for a wide variety of small or high-throughput experiments such as RNAi screens, microarrays, massive sequencing, immunoblot or in situ hybridization, among others. Because of its transparency, C. elegans structures can be distinguished under the microscope using Differential Interference Contrast microscopy, also known as Nomarski microscopy. The use of a fluorescent DNA binder, DAPI (4',6-diamidino-2-phenylindole), for instance, can lead to the specific identification and localization of individual cells, as well as subcellular structures/defects associated to them.