Basic Caenorhabditis Elegans Methods: Synchronization And Observation

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 (1). 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 (2). Discoveries in this nematode have broad implications because many cellular and molecular processes that control animal development are evolutionary conserved (3). 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 (4,5) together with the deep annotation of its genome turn this nematode into a great model in fields as diverse as the neurobiology (6), aging (7,8), stem cell biology (9) and germ line biology (10). 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

RSR-2, the Caenorhabditis elegans Ortholog of Human Spliceosomal Component SRm300/SRRM2, Regulates Development by Influencing the Transcriptional Machinery

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

Cell therapy with hiPSC-derived RPE cells and RPCs prevents visual function loss in a rat model of retinal degeneration

Photoreceptor loss is the principal cause of blindness in retinal degenerative diseases (RDDs). Whereas some therapies exist for early stages of RDDs, no effective treatment is currently available for later stages, and once photoreceptors are lost, the only option to rescue vision is cell transplantation. With the use of the Royal College of Surgeons (RCS) rat model of retinal degeneration, we sought to determine whether combined transplantation of human-induced pluripotent stem cell (hiPSC)-derived retinal precursor cells (RPCs) and retinal pigment epithelial (RPE) cells was superior to RPE or RPC transplantation alone in preserving retinal from degeneration. hiPSC-derived RPCs and RPE cells expressing (GFP) were transplanted into the subretinal space of rats. In vivo monitoring showed that grafted cells survived 12 weeks in the subretinal space, and rats treated with RPE + RPC therapy exhibited better conservation of the outer nuclear layer (ONL) and visual response than RPE-treated or RPC-treated rats. Transplanted RPE cells integrated in the host RPE layer, whereas RPC mostly remained in the subretinal space, although a limited number of cells integrated in the ONL. In conclusion, the combined transplantation of hiPSC-derived RPE and RPCs is a potentially superior therapeutic approach to protect retina from degeneration in RDDs

Modeling of autosomal-dominant retinitis pigmentosa in Caenorhabditis elegans uncovers a nexus between global impaired functioning of certain splicing factors and cell type-specific apoptosis

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

Comparative study of human embryonic stem cells (hESC) and human induced pluripotent stem cells (hiPSC) as a treatment for retinal dystrophies

Retinal dystrophies (RD) are major causes of familial blindness and are characterized by progressive dysfunction of photoreceptor and/or retinal pigment epithelium (RPE) cells. In this study, we aimed to evaluate and compare the therapeutic effects of two pluripotent stem cell (PSC)-based therapies. We differentiated RPE from human embryonic stem cells (hESCs) or human-induced pluripotent stem cells (hiPSCs) and transplanted them into the subretinal space of the Royal College of Surgeons (RCS) rat. Once differentiated, cells from either source of PSC resembled mature RPE in their morphology and gene expression profile. Following transplantation, both hESC- and hiPSC-derived cells maintained the expression of specific RPE markers, lost their proliferative capacity, established tight junctions, and were able to perform phagocytosis of photoreceptor outer segments. Remarkably, grafted areas showed increased numbers of photoreceptor nuclei and outer segment disk membranes. Regardless of the cell source, human transplants protected retina from cell apoptosis, glial stress and accumulation of autofluorescence, and responded better to light stimuli. Altogether, our results show that hESC- and hiPSC-derived cells survived, migrated, integrated, and functioned as RPE in the RCS rat retina, providing preclinical evidence that either PSC source could be of potential benefit for treating RD

Rapid evolution of coordinated and collective movement in response to artificial selection.

Collective motion occurs when individuals use social interaction rules to respond to the movements and positions of their neighbors. How readily these social decisions are shaped by selection remains unknown. Through artificial selection on fish (guppies, Poecilia reticulata) for increased group polarization, we demonstrate rapid evolution in how individuals use social interaction rules. Within only three generations, groups of polarization-selected females showed a 15% increase in polarization, coupled with increased cohesiveness, compared to fish from control lines. Although lines did not differ in their physical swimming ability or exploratory behavior, polarization-selected fish adopted faster speeds, particularly in social contexts, and showed stronger alignment and attraction responses to multiple neighbors. Our results reveal the social interaction rules that change when collective behavior evolves

A comprehensive functional study of Caenorhabditis elegans rsr-2 uncovers a new link between splicing and transcription

Durant el transcurs d’un rastreig a gran escala per RNA d’interferència (iARN) en Caenorhabditis elegans, el gen rsr-2 va ser identificat com a interactor genètic del gen lin-35 Retinoblastoma, l’homòleg de la família humana de Retinoblastoma. El gen rsr-2 és l’ortòleg de la proteina humana d’splicing SRm300/SRRM2. A diferència del seu ortòleg en llevat Cwc21, rsr-2 és essencial per la viabilitat. Degut a que la forta inactivació d’rsr-2 produeix uns fenotips molt severs, hem aprofitat l’efecte lleu que produeix l’ARN interferent a través de l’aliment per estudiar les funcions d’rsr-2 durant el desenvolupament. Els assatjos d’iARN d’rsr-2 juntament amb anàlisis d’epistàsia genètica situen rsr-2 en la via de la determinació sexual de la línia germinal. Tot i així, tiling arrays d’animals rsr-2(iARN) no han revelat defectes significatius en l’splicing. Gràcies a experiments d’immunofluorescència, hem observat que un anticòs específic per RSR-2 co-localitza amb cromatina en nuclis de cèl·lules de la línia germinal. Interessantment, experiments d’immunoprecipitació de cromatina i seqüenciació massiva (ChIP-Seq) han desvetllat que RSR-2 co-precipita cromatina seguint un patró similar al de l’ARN polimerassa II. Aquests experiments de ChIP-Seq també han fet palès que RSR-2 és reclutada a la cromatina d’un mode que és independent de l’splicing i suggereixen que RSR-2 podria desenvolupar un rol de regulador transcripcional. Addicionalment, hem explorat els transcriptomes d’animals rsr-2(iARN) i prp-8(iARN) en estadi de larva 3 (L3), fet que ens ha permès classificar rsr-2 com a un factor d’splicing no essencial. Conjuntament, el nostre estudi mostra que RSR-2 és una proteina multifuncional que regula el desenvolupament de Caenorhabditis elegans influenciant, i probablement acoblant, els processos d’splicing i transcripció.During the course of a large scale interference RNA (RNAi) screen in Caenorhabditis elegans, rsr-2 was identified as a genetic interactor of lin-35 Rb, the homolog of human Retinoblastoma. The rsr-2 gene encodes the ortholog of the human spliceosomal protein SRm300/SRRM2. In contrast to its yeast ortholog Cwc21, rsr-2 is essential for viability. Since strong inactivation of rsr-2 produces severe phenotypes, we took advantage of the mild effect of RNAi by feeding to study functions of rsr-2 during development. rsr-2 RNAi assays and genetic epistasis analysis locate rsr-2 within the germ line sex determination pathway but tiling arrays of rsr-2(RNAi) animals do not disclose significant splicing defects. By inmunofluorescence, we observe that an antibody specific for RSR-2 co-localizes with chromatin in germ line nuclei. Interestingly, ChIP-Seq experiments reveal that RSR-2 co-precipitates chromatin in a pattern similar to that of RNA Polymerase II. These ChIP-Seq assays also evidenced a splicing-independent recruitment of RSR-2 to chromatin and suggest that RSR-2 could have a role in regulating transcription. Moreover, we have explored the transcriptomes of rsr-2(RNAi) and prp-8(RNAi) L3 worms by RNA-Seq, which classified rsr-2 as a non-essential splicing factor. Altogether, our study shows that RSR-2 is a multifunctional protein that regulates development by influencing, and probably coupling, splicing and transcription

Neovascular Progression and Retinal Dysfunction in the Laser-Induced Choroidal Neovascularization Mouse Model

The mouse model of laser-induced choroidal neovascularization (LI-CNV) has been widely used to study neovascular age-related macular degeneration; however, it still lacks a comprehensive characterization. Here, CNV was induced in the eyes of 12-week-old C57BL/6J male mice by argon laser irradiation. We studied the CNV lesion progression of an LI-CNV mouse cohort by using multimodal imaging (color fundus, optical coherence tomography (OCT), and fluorescence angiography, focal electroretinography features for 14 days, and related cytokines, angiogenic factors, and reactive gliosis for 5 days. CNV lesions involving the rupture of the Bruch’s membrane were confirmed using funduscopy and OCT after laser photocoagulation. During the initial stage, from the CNV induction until day 7, CNV lesions presented leakage observed by using fluorescence angiography and a typical hyperreflective area with cell infiltration, subretinal leakage, and degeneration of photoreceptors observed through OCT. This correlated with decreased retinal responses to light. Moreover, inflammatory and angiogenic markers were reduced to basal levels in the first 5 days of CNV progression. In contrast, reactive gliosis and the VEGF expression in retinal sections were sustained, with infiltration of endothelial cells in the subretinal space. In the second stage, between days 7 and 14 post-induction, we observed stabilization of the CNV lesions, a hyperfluorescent area corresponding to the formation of fibrosis, and a partial rescue of retinal function. These findings suggest that the LI-CNV lesion development goes through an acute phase during the first seven days following induction, and then the CNV lesion stabilizes. According to these results, this model is suitable for screening anti-inflammatory and anti-angiogenic drugs in the early stages of LI-CNV. At the same time, it is more convenient for screening anti-fibrotic compounds in the later stages

A comprehensive functional study of Caenorhabditis elegans rsr-2 uncovers a new link between splicing and transcription

Durant el transcurs d'un rastreig a gran escala per RNA d'interferència (iARN) en Caenorhabditis elegans, el gen rsr-2 va ser identificat com a interactor genètic del gen lin-35 Retinoblastoma, l'homòleg de la família humana de Retinoblastoma. El gen rsr-2 és l'ortòleg de la proteina humana d'splicing SRm300/SRRM2. A diferència del seu ortòleg en llevat Cwc21, rsr-2 és essencial per la viabilitat. Degut a que la forta inactivació d'rsr-2 produeix uns fenotips molt severs, hem aprofitat l'efecte lleu que produeix l'ARN interferent a través de l'aliment per estudiar les funcions d'rsr-2 durant el desenvolupament. Els assatjos d'iARN d'rsr-2 juntament amb anàlisis d'epistàsia genètica situen rsr-2 en la via de la determinació sexual de la línia germinal. Tot i així, tiling arrays d'animals rsr-2(iARN) no han revelat defectes significatius en l'splicing. Gràcies a experiments d'immunofluorescència, hem observat que un anticòs específic per RSR-2 co-localitza amb cromatina en nuclis de cèl·lules de la línia germinal. Interessantment, experiments d'immunoprecipitació de cromatina i seqüenciació massiva (ChIP-Seq) han desvetllat que RSR-2 co-precipita cromatina seguint un patró similar al de l'ARN polimerassa II. Aquests experiments de ChIP-Seq també han fet palès que RSR-2 és reclutada a la cromatina d'un mode que és independent de l'splicing i suggereixen que RSR-2 podria desenvolupar un rol de regulador transcripcional. Addicionalment, hem explorat els transcriptomes d'animals rsr-2(iARN) i prp-8(iARN) en estadi de larva 3 (L3), fet que ens ha permès classificar rsr-2 com a un factor d'splicing no essencial. Conjuntament, el nostre estudi mostra que RSR-2 és una proteina multifuncional que regula el desenvolupament de Caenorhabditis elegans influenciant, i probablement acoblant, els processos d'splicing i transcripció.During the course of a large scale interference RNA (RNAi) screen in Caenorhabditis elegans, rsr-2 was identified as a genetic interactor of lin-35 Rb, the homolog of human Retinoblastoma. The rsr-2 gene encodes the ortholog of the human spliceosomal protein SRm300/SRRM2. In contrast to its yeast ortholog Cwc21, rsr-2 is essential for viability. Since strong inactivation of rsr-2 produces severe phenotypes, we took advantage of the mild effect of RNAi by feeding to study functions of rsr-2 during development. rsr-2 RNAi assays and genetic epistasis analysis locate rsr-2 within the germ line sex determination pathway but tiling arrays of rsr-2(RNAi) animals do not disclose significant splicing defects. By inmunofluorescence, we observe that an antibody specific for RSR-2 co-localizes with chromatin in germ line nuclei. Interestingly, ChIP-Seq experiments reveal that RSR-2 co-precipitates chromatin in a pattern similar to that of RNA Polymerase II. These ChIP-Seq assays also evidenced a splicing-independent recruitment of RSR-2 to chromatin and suggest that RSR-2 could have a role in regulating transcription. Moreover, we have explored the transcriptomes of rsr-2(RNAi) and prp-8(RNAi) L3 worms by RNA-Seq, which classified rsr-2 as a non-essential splicing factor. Altogether, our study shows that RSR-2 is a multifunctional protein that regulates development by influencing, and probably coupling, splicing and transcription