RESUMEN
Germ cells contain non-membrane bound cytoplasmic organelles that help maintain germline integrity. In C. elegans they are called P granules; without them, the germline undergoes partial masculinization and aberrant differentiation. One key P-granule component is the Argonaute CSR-1, a small-RNA binding protein that antagonizes accumulation of sperm-specific transcripts in developing oocytes and fine-tunes expression of proteins critical to early embryogenesis. Loss of CSR-1 complex components results in a very specific, enlarged P-granule phenotype. In a forward screen to identify mutants with abnormal P granules, ten alleles were recovered with a csr-1 P-granule phenotype, eight of which contain mutations in known components of the CSR-1 complex (csr-1, ego-1, ekl-1, and drh-3). The remaining two alleles are in a novel gene now called elli-1 (enlarged germline granules). ELLI-1 is first expressed in primordial germ cells during mid-embryogenesis, and continues to be expressed in the adult germline. While ELLI-1 forms cytoplasmic aggregates, they occasionally dock, but do not co-localize with P granules. Instead, the majority of ELLI-1 aggregates accumulate in the shared germline cytoplasm. In elli-1 mutants, several genes that promote RNAi and P-granule accumulation are upregulated, and embryonic lethality, sterility, and RNAi resistance in a hypomorphic drh-3 allele is enhanced, suggesting that ELLI-1 functions with CSR-1 to modulate RNAi activity, P-granule accumulation, and post-transcriptional expression in the germline.
Asunto(s)
Proteínas de Caenorhabditis elegans/genética , Caenorhabditis elegans/genética , Gránulos Citoplasmáticos/metabolismo , Células Germinativas/metabolismo , Interferencia de ARN , Factores Generales de Transcripción/genética , Alelos , Animales , Animales Modificados Genéticamente , Secuencia de Bases , Caenorhabditis elegans/embriología , Caenorhabditis elegans/crecimiento & desarrollo , Proteínas de Caenorhabditis elegans/metabolismo , ARN Helicasas DEAD-box/genética , ARN Helicasas DEAD-box/metabolismo , Perfilación de la Expresión Génica/métodos , Regulación del Desarrollo de la Expresión Génica , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Inmunohistoquímica , Hibridación Fluorescente in Situ , Microscopía Fluorescente , Mutación , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , ARN Polimerasa Dependiente del ARN/genética , ARN Polimerasa Dependiente del ARN/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores Generales de Transcripción/metabolismoRESUMEN
Background: Intervertebral disc (IVD) degeneration is a major contributor to back pain and disability. The cause of IVD degeneration is multifactorial, with no disease-modifying treatments. Mouse models are commonly used to study IVD degeneration; however, the effects of anatomical location, strain, and sex on the progression of age-associated degeneration are poorly understood. Methods: A longitudinal study was conducted to characterize age-, anatomical-, and sex-specific differences in IVD degeneration in two commonly used strains of mice, C57BL/6 and CD-1. Histopathological evaluation of the cervical, thoracic, lumbar, and caudal regions of mice at 6, 12, 20, and 24 months of age was conducted by two blinded observers at each IVD for the nucleus pulposus (NP), annulus fibrosus (AF), and the NP/AF boundary compartments, enabling analysis of scores by tissue compartment, summed scores for each IVD, or averaged scores for each anatomical region. Results: C57BL/6 mice displayed mild IVD degeneration until 24 months of age; at this point, the lumbar spine demonstrated the most degeneration compared to other regions. Degeneration was detected earlier in the CD-1 mice (20 months of age) in both the thoracic and lumbar spine. In CD-1 mice, moderate to severe degeneration was noted in the cervical spine at all time points assessed. In both strains, age-associated IVD degeneration in the thoracic and lumbar spine was associated with increased histopathological scores in all IVD compartments. In both strains, minimal degeneration was detected in caudal IVDs out to 24 months of age. Both C57BL/6 and CD-1 mice displayed sex-specific differences in the presentation and progression of age-associated IVD degeneration. Conclusions: These results showed that the progression and severity of age-associated degeneration in mouse models is associated with marked differences based on anatomical region, sex, and strain. This information provides a fundamental baseline characterization for users of mouse models to enable effective and appropriate experimental design, interpretation, and comparison between studies.
RESUMEN
In Caenorhabditis elegans, germline expression programs are actively repressed in somatic tissue by components of the synMuv (synthetic multi-vulva) B chromatin remodeling complex, which include homologs of tumor suppressors Retinoblastoma (Rb/LIN-35) and Malignant Brain Tumor (MBT/LIN-61). However, the full scope of pathways that suppress germline expression in the soma is unknown. To address this, we performed a mutagenesis and screened for somatic expression of GFP-tagged PGL-1, a core P-granule nucleating protein. Eight alleles were isolated from 4000 haploid genomes. Five of these alleles exhibit a synMuv phenotype, whereas the remaining three were identified as hypomorphic alleles of known synMuv B genes, lin-13 and dpl-1. These findings suggest that most suppressors of germline programs in the soma of C. elegans are either required for viability or function through synMuv B chromatin regulation.
Asunto(s)
Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Alelos , Animales , Animales Modificados Genéticamente , Mapeo Cromosómico , Expresión Génica , Genes Reporteros , Células Germinativas , Mutación , Interferencia de ARN , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismoRESUMEN
In North America, tick-borne relapsing fever of humans is most frequently caused by infection with the spirochete Borrelia hermsii. Prior to our investigation, this spirochete was not known to infect dogs although another species, Borrelia turicatae, has been isolated from domestic canids in Florida and Texas. A clinically ill dog in Washington, USA, was spirochetemic upon examination. Spirochetes were isolated from the dog's serum and examined by PCR and multi-locus sequence typing. DNA sequences for 7 loci all typed the spirochete as B. hermsii and a member of genomic group II of this species. Therefore, companion dogs that reside in rustic cabins in higher elevation forests are at risk of infection with B. hermsii.