Oct4 Is a Key Regulator of Vertebrate Trunk Length Diversity

The deposited article version is a post-print version (final draft post-refereeing) and included supplementary information.Vertebrates exhibit a remarkably broad variation in trunk and tail lengths. However, the evolutionary and developmental origins of this diversity remain largely unknown. Posterior Hox genes were proposed to be major players in trunk length diversification in vertebrates, but functional studies have so far failed to support this view. Here we identify the pluripotency factor Oct4 as a key regulator of trunk length in vertebrate embryos. Maintaining high Oct4 levels in axial progenitors throughout development was sufficient to extend trunk length in mouse embryos. Oct4 also shifted posterior Hox gene-expression boundaries in the extended trunks, thus providing a link between activation of these genes and the transition to tail development. Furthermore, we show that the exceptionally long trunks of snakes are likely to result from heterochronic changes in Oct4 activity during body axis extension, which may have derived from differential genomic rearrangements at the Oct4 locus during vertebrate evolution.Fundação para a Ciência e a Tecnologia grants: ( PTDC/BEX-BID/0899/2014, SFRH/BD/51876/2012); Santa Casa da Misericordia de Lisboa grant: (SCML-MC-60-2014); Howard Hughes Medical; HHMI international graduate student research fellowship.info:eu-repo/semantics/publishedVersio

Gene function in schistosomes: recent advances toward a cure

Made available in DSpace on 2015-08-19T13:49:24Z (GMT). No. of bitstreams: 2 license.txt: 1914 bytes, checksum: 7d48279ffeed55da8dfe2f8e81f3b81f (MD5) arnon_jurbrg_brindley_IOC_215.pdf: 402241 bytes, checksum: f019c3df8c3bd47305d3846a9ededf40 (MD5) Previous issue date: 2015Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Pesquisa do Timo. Rio de Janeiro, RJ, Brasil / Universidade Federal do Rio de Janeiro. Instituto de Ciências Biomédicas. Programa de Pós-Graduação em Biologia Celular e do Desenvolvimento. Rio de Janeiro, RJ, Brasil.School of Medicine and Health Sciences. Research Center for Neglected Diseases of Poverty. Department of Microbiology, Immunology and Tropical Medicine. Washington, DC, USA

The Role of Embryonic Chick Muscle Cell Culture in the Study of Skeletal Myogenesis

The mechanisms involved in the development of skeletal muscle fibers have been studied in the last 70 years and yet many aspects of this process are still not completely understood. A myriad of in vivo and in vitro invertebrate and vertebrate animal models has been used for dissecting the molecular and cellular events involved in muscle formation. Among the most used animal models for the study of myogenesis are the rodents rat and mouse, the fruit fly Drosophila, and the birds chicken and quail. Here, we describe the robustness and advantages of the chick primary muscle culture model for the study of skeletal myogenesis. In the myoblast culture obtained from embryonic chick pectoralis muscle it is possible to analyze all the steps involved in skeletal myogenesis, such as myoblast proliferation, withdrawal from cell cycle, cell elongation and migration, myoblast alignment and fusion, the assembly of striated myofibrils, and the formation of multinucleated myotubes. The fact that in vitro chick myotubes can harbor hundreds of nuclei, whereas myotubes from cell lines have only a dozen nuclei demonstrates the high level of differentiation of the autonomous chick myogenic program. This striking differentiation is independent of serum withdrawal, which points to the power of the model. We also review the major pro-myogenic and anti-myogenic molecules and signaling pathways involved in chick myogenesis, in addition to providing a detailed protocol for the preparation of embryonic chick myogenic cultures. Moreover, we performed a bibliometric analysis of the articles that used this model to evaluate which were the main explored topics of interest and their contributors. We expect that by describing the major findings, and their advantages, of the studies using the embryonic chick myogenic model we will foster new studies on the molecular and cellular process involved in muscle proliferation and differentiation that are more similar to the actual in vivo condition than the muscle cell lines.</jats:p

Use of a saline gradient for the diagnosis of schistosomiasis

The development of novel methods for parasitological diagnosis that are both highly sensitive and low in cost has been strongly recommended by the World Health Organization. In this study, a new technique for diagnosis of schistosomiasis mansoni is proposed based on the differential sedimentation of eggs when subjected to a slow continuous flux of 3% saline solution through a porous plaque. This influx suspends low-density faecal material, effectively cleaning the sample. The remaining sediment covering the porous plaque surface is then transferred to a glass slide and examined under a bright field microscope. Twelve Kato-Katz slides were used for comparison in the present study. Our results suggest that the saline gradient method detects a signifi-cantly higher number of eggs than the 12 Kato-Katz slides (p < 0.0001). We also found microscopic inspection to be quicker and easier with our newly described method. After cleaning the sample, the obtained sediment can also be conserved in a 10% formaldehyde solution and examined for at least 45 days later without statistically significant egg count differences

Use of a saline gradient for the diagnosis of schistosomiasis

The development of novel methods for parasitological diagnosis that are both highly sensitive and low in cost has been strongly recommended by the World Health Organization. In this study, a new technique for diagnosis of schistosomiasis mansoni is proposed based on the differential sedimentation of eggs when subjected to a slow continuous flux of 3% saline solution through a porous plaque. This influx suspends low-density faecal material, effectively cleaning the sample. The remaining sediment covering the porous plaque surface is then transferred to a glass slide and examined under a bright field microscope. Twelve Kato-Katz slides were used for comparison in the present study. Our results suggest that the saline gradient method detects a signifi-cantly higher number of eggs than the 12 Kato-Katz slides (p < 0.0001). We also found microscopic inspection to be quicker and easier with our newly described method. After cleaning the sample, the obtained sediment can also be conserved in a 10% formaldehyde solution and examined for at least 45 days later without statistically significant egg count differences

A new miracidia hatching device for diagnosing schistosomiasis

It is still imperative to develop a parasitological technique highly sensitive for diagnosing schistosomiasis in epidemiological and individual surveys. A simple and cheap hatching device with a collecting container was manufactured and tested under experimental conditions. Twelve Kato-Katz slides were performed as golden standard for comparison. Quantitative results can be carried out by counting miracidia in a plate and parasite load can be calculated (miracidia/gram of feces). Statistically significant values were higher in the hatching test. More sensitive results, with statistical significance, were achieved using 1.5 g of feces (which corresponds to 36 Kato-Katz slides) than by using the Kato-Katz method. Advantages of this technique and its limitations are presented

Genome Editing for Functional Genomics of Schistosomes

The Streptococcus pyogenes Type II CRISPR system is the keystone of the CRISPR revolution. The system centers on a programmable endonuclease that catalyzes a double stranded break (DSB) in target DNA. The system has been shown to be active in many species including human, mouse, zebra fish, fruit fly, malarial parasite and yeast. It has revolutionized experimental genome editing, and portends hitherto unparalleled advances and positive prospects for gene therapy, biomedicine, and biological systems at large. Adaption of CRISPR technology for editing the genome of schistosomes and other parasitic platyhelminths would be desirable. Here we targeted the IPSE gene of Schistosoma mansoni for ‘knockout’ - deletion mutation in the coding region of the gene. First, using a double reporter plasmid system, NIH 3T3 fibroblasts were transfected with pX330-IPSE1 and pRGS-tgt-IPSE1. By FACS, ~9% cells were RFP+ve, GFP+ve, indicating cleavage of exon 1 of SmIPSE gene (within pRGS-tgt-IPSE1). Second, in vitro incubation of plasmid pRGS-tgt-SmIPSE1 that includes part of exon 1 of the IPSE gene, including a protospacer adjacent motif (PAM), with a macromolecular complex of guide RNA (gRNA) and recombinant Cas9 linearized the plasmid, presumably the consequence of a directed DSB catalyzed by Cas9. Third, cultured schistosomula were transfected using square wave electroporation with recombinant Cas9 of S. pyogenes complexed with gRNA matching residues 22 - 44 of exon 1 of the IPSE gene. Indels at the IPSE locus were evident by two hours later, detected by quantitative PCR, in ~13% of the cells of the parasites. The Type II Cas9 System is active in schistosomes